Succinimide-activated nitroxyl compounds and methods for the use thereof for nitroxylation of proteins

ABSTRACT

The present invention relates to succinimide-activated nitroxyl compounds and methods for the synthesis of such compounds. The present invention also relates to the use of succinimide-activated nitroxyl compounds to prepare nitroxylated proteins, for example nitroxylated heme proteins (e.g., nitroxylated hemoglobin and nitroxylated myoglobin). The nitroxylated proteins are optionally also conjugated to a polyalkylene oxide (PAO), for example to a polyethylene glycol (PEG). Polynitroxylated heme proteins are useful as oxygen therapeutic agents (OTAs). The invention further relates to pharmaceutical compositions of the nitroxylated proteins and methods for the use of nitroxylated proteins in the treatment of various conditions.

FIELD OF THE INVENTION

The present invention generally relates to succinimide-activatednitroxyl compounds and methods for the synthesis ofsucccinimide-activated nitroxyl compounds. The present invention alsorelates to the use of succinimide-activated nitroxyl compounds toprepare nitroxylated proteins, for example nitroxylated heme proteins(e.g., nitroxylated hemoglobin and nitroxylated myoglobin). Thenitroxylated proteins are optionally also conjugated to a polyalkyleneoxide (PAO), for example to a polyethylene glycol (PEG).Polynitroxylated heme proteins are useful as oxygen therapeutic agents(OTAs) and are capable of delivering molecular oxygen, carbon monoxide,nitric oxide and mixtures thereof. Thus, the invention further includespharmaceutical compositions of the nitroxylated proteins and methods forthe use of nitroxylated proteins in the treatment of various conditions.

BACKGROUND OF THE INVENTION

Hemoglobin-based oxygen carriers (“HBOC”) have long been associated withvasoconstriction that has been attributed to nitric oxide (NO)scavenging by heme. Oxygen carriers that are useful as oxygentherapeutics (sometimes referred to as “oxygen-carrying plasmaexpanders”), such as stabilized hemoglobin (Hb), have been shown to havelimited efficacy because they scavenge nitric oxide, causingvasoconstriction and hypertension. The propensity of these oxygencarrying solutions to cause vasoconstriction can manifest ashypertension in animals and man. Although the mechanisms underlying thevasoconstrictive effects of HBOCs are not well understood, it has beensuggested that the heme iron may combine rapidly and irreversibly withendogenous NO, a powerful vasodilator, thereby causing vasoconstriction.

In part because of these vasoconstrictive effects, no oxygen carrier todate has been entirely successful as an oxygen therapeutic agent (OTA),although products comprising modified cell-free Hb have been the mostpromising. Human Hb cross-linked between α-chains withbis-dibromosalicyl-fumarate (ααHb) was developed by the U.S. Army as amodel red cell substitute, but was abandoned after it exhibited severeincreases in pulmonary and systemic vascular resistance (Hess, J. etal., 1991, Blood 78:356A). A commercial version of this product was alsoabandoned after a disappointing Phase III clinical trial (Winslow, R.M., 2000, Vox Sang 79:1-20).

Two molecular approaches have been advanced in attempting to overcomethe NO binding activity of Hb. The first approach used site-directedmutagenesis of the distal heme pocket in an attempt to create arecombinant hemoglobin with reduced NO-binding affinity (Eich, R.F. etal., 1996, Biochem. 35:6976-83). The second approach used a chemicalmodification approach wherein the size of the Hb was enhanced througholigomerization in an attempt to reduce or possibly completely inhibitthe extravasation of Hb from the vascular space into the interstitialspace (Hess, J. R. et al., 1978, J. Appl. Physiol. 74:1769-78; Muldoon,S. M. et al., 1996, J. Lab. Clin. Med. 128:579-83; Macdonald, V. W. etal., 1994, Biotechnology 22:565-75; Furchgott, R., 1984, Ann. Rev.Pharmacol. 24:175-97; and Kilbourne, R. et al., 1994, Biochem. Biophys.Res. Commun 199:155-62).

In fact, recombinant Hbs with reduced association binding rates for NOhave been produced that are less hypertensive in top-load ratexperiments (Doherty, D. H. etg al. 1998, Nature Biotechnology16:672-676 and Lemon, D. D. et al. 1996, Biotech 24:378). However,studies suggest that NO binding may not be the only explanation for thevasoactivity of Hb. It has been found that certain large Hb molecules,such as those modified with polyethylene glycol (PEG), were virtuallyfree of vasoconstriction, even though their NO association rates wereidentical to those of the severely hypertensive ααHb (Rohlfs, R. J. etal. 1998, J Biol. Chem. 273:12128-12134). Furthermore, it was found thatPEG-Hb was extraordinarily effective in preventing the consequences ofhemorrhage when given as an exchange transfusion prior to hemorrhage(Winslow, R. M. et al. 1998, J. Appl. Physiol. 85:993-1003).

The conjugation of PEG to Hb reduces its antigenicity and extends itscirculation half-life. However, the PEG conjugation reaction has beenreported to result in dissociation of Hb tetramers into αβ-dimersubunits causing gross hemoglobinuria in exchange-transfused ratsreceiving PEG-conjugates of Hb monomeric units below 40,000 Daltons(“Da”) (Iwashita and Ajisaka Organ-Directed Toxicity: Chem. IndiciesMech., Proc. Symp., Brown et al. 1981, Eds. Pergamon, Oxford, Englandpgs 97-101). A polyalkylene oxide (“PAO”) conjugated Hb having amolecular weight greater than 84,000 Daltons was prepared by Enzon, Inc.(U.S. Pat. No. 5,650,388) that carried about 10 copies of PEG-5,000chains linked to Hb at its a and ε-amino groups. This degree ofsubstitution was described as avoiding clinically significantnephrotoxicity associated with hemoglobinuria in mammals. However, theconjugation reaction resulted in a heterogeneous conjugate populationand contained other undesirable reactants that had to be removed bycolumn chromatography.

PEG conjugation is typically carried out through the reaction of anactivated PEG moiety with a functional group on the surface ofbiomolecules. The most common functional groups are the amino groups oflysine, imidazole groups of histidine residues, and the N-terminus ofproteins; thiol groups of cysteine residues; and the hydroxyl groups ofserine, threonine and tyrosine residues and the C-terminus of theprotein. PEG is usually activated by converting the hydroxyl terminus toa reactive moiety capable of reacting with these functional groups in amild aqueous environment. One of the most common monofunctional PEGsused for conjugation of therapeutic biopharmaceuticals is methoxy-PEG(“mPEG-OH”), which has only one functional group (i.e. hydroxyl), thusminimizing cross-linking and aggregation problems that are associatedwith bifunctional PEG. However, mPEG-OH is often contaminated with highmolecular weight bifunctional PEG (i.e. “PEG diol”), which can range ashigh as 10 to 15% (Dust J. M. et al. 1990, Macromolecule 23:3742-3746)due to its production process. This bifunctional PEG diol has roughlytwice the size of the desired monofunctional PEG. The contaminationproblem is further aggravated as the molecular weight of PEG increases.The purity of mPEG-OH is especially critical for the production ofPEGylated biotherapeutics, because the FDA requires a high level ofreproducibility in the production processes and quality of the finaldrug product.

Conjugation of Hb to PAOs has been performed in both the oxygenated anddeoxygenated states. U.S. Pat. No. 6,844,317 describes conjugating Hb inthe oxygenated, or “R” state by equilibrating Hb with the atmosphereprior to conjugation to enhance the oxygen affinity of the resultantPEG-Hb conjugate. Others describe a deoxygenation step prior toconjugation to diminish the oxygen affinity and increase structuralstability, enabling the Hb to withstand the physical stresses ofchemical modification, diafiltration and/or sterile filtration andpasteurization (U.S. Pat. No. 5,234,903). For intramolecularcross-linking of Hb, it is suggested that deoxygenating Hb prior tomodification may be required to expose lysine 99 of the α-chain to thecross-linking reagent (U.S. Pat. No. 5,234,903).

The kinetics of Hb thiolation with 2-iminothiolane prior to conjugationwith PEG was investigated by Acharya et al. (U.S. Pat. No. 7,501,499).It was observed that increasing the concentration of iminothiolane from10-fold, which introduced an average of five extrinsic thiols pertetramer, to 30-fold nearly doubled the number of extrinsic thiols onHb. However, the size enhancement seen after PEG conjugation was onlymarginal, even with double the number of thiols. This suggested that theconjugation reaction in the presence of 20-fold molar excess ofmaleimidyl PEG-5000 covered the surface of the Hb with less reactivethiols, resulting in steric interference that resisted furthermodification of Hb with more reactive thiols. Consequently, to achievethe desired degree of conjugation of modified Hb (i.e. 6±1 PEG per Hbmolecule), Acharya et al. thiolated Hb with an 8-15 molar excess ofiminothiolane, and then reacted the thiolated Hb with a 16-30 fold molarexcess of maleimidyl PEG-5000. However, these high molar excess reactantconcentrations in large-scale production significantly increase the costfor preparing the HBOC and increase the heterogeneity of the finalproduct. Moreover, such high molar excess of the maleimidyl PEG-5000also results in a more heterogeneous product with the production of agreater number of unwanted side reactants.

In previous studies, it was observed that the molecular size of surfacemodified hemoglobin has to be large enough to avoid being cleared by thekidneys and to achieve the desired circulation half-life. Blumenstein,J. et al., determined that this could be achieved at, or above, amolecular weight of 84,000 Daltons (“Da”) (“Blood Substitutes and PlasmaExpanders,” Alan R. Liss, editors, New York, N.Y., pages 205-212(1978)). In that study, the authors conjugated dextran of varyingmolecular weight to Hb. They reported that a conjugate of Hb (with amolecular weight of 64,000 Da) and dextran (having a molecular weight of20,000 Da) “was cleared slowly from the circulation and negligiblythrough the kidneys.” Further, it was observed that increasing themolecular weight above 84,000 Da did not significantly alter theseclearance curves. Intramolecular cross-linking chemically binds togethersubunits of the tetrameric hemoglobin unit to prevent the formation ofdimers which are prematurely excreted by the kidney. (See, e.g., U.S.Pat. No. 5,296,465)

Nitroxides are well-established antioxidant compounds of low toxicitythat attenuate oxidative damage in animal models of inflammatory diseaseand preserve bioavailable NO gas. They are believed to exert protectiveeffects principally by acting as SOD mimetics or radical scavengers.Thus, polynitroxylated compounds have antioxidant and anti-inflammatoryproperties. This is not to be confused with combining HBOCs with nitricoxide (NO) donor molecules, which have been reported to enhance vascularrelaxation. See, e.g., U.S. Patent Application Publication No.2010/0311657. However, SOD-mimetic nitroxides have a short plasmahalf-life due to their small size, and thus it is difficult to maintainthe antioxidant efficacy of these molecules in vivo.

In view of the foregoing, there is a need in the art for oxygentherapeutic agents that do not cause vasoconstriction and hypertensionand that have antioxidant and anti-inflammatory properties.

Moreover, current methods for activating nitroxides for use asnitroxylating agents are multistep and expensive processes. There istherefore a need in the art for methods for simple and inexpensivemethods for making activated nitroxide compounds for use asnitroxylating agents.

SUMMARY OF THE INVENTION

One aspect of the present invention relates to a nitroxylating agent offormula (I):

wherein each of R₁, R₂, R₃, and R₄ is independently C₁-C₄ alkyl; X isoxygen, sulfur, nitrogen, phosphorus, or silicon; Y is CH₂; n is 0 or 1;and m is 0 or 1.

Another aspect of the invention relates to a method for preparing anitroxylating agent of the formula (II):

comprising reacting a compound having the formula (III)

with N,N′-disuccinimidyl carbonate (DSC) in the presence of an organicbase; wherein each of R₁, R₂, R₃, and R₄ is independently C₁-C₄ alkyl; Xis oxygen, sulfur, nitrogen, phosphorus, or silicon; Y is CH₂; and m is0 or 1.

Yet another aspect of the invention is also directed to a method forpreparing a nitroxylating agent of the formula (IV):

comprising reacting a compound having the formula (V)

with N-hydroxysuccinimide (NHS) in the presence ofN,N′-dicyclohexylcarbodiimide (DCC); wherein each of R₁, R₂, R₃, and R₄is independently C₁-C₄ alkyl; Y is CH₂; and m is 0 or 1.

A further aspect is a nitroxylated protein comprising at least onenitroxylated amino group and the nitroxylated protein can have thestructure (VI):

wherein Z represents the protein; each of R₁, R₂, R₃, and R₄ areindependently C₁-C₄ alkyl; X is oxygen, sulfur, nitrogen, phosphorus, orsilicon; Y is CH₂; m is 0 or 1; n is the average number of activated-PEGpolymers conjugated to the protein, the —NH— group is an amine group ofthe protein and N is a nitrogen of the protein.

Yet another aspect or the invention is a nitroxylated protein that cancomprise at least one nitroxylated amino group, the nitroxylated proteinhaving the structure (VII):

wherein Z represents the protein; each of R₁, R₂, R₃, and R₄ areindependently C₁-C₄ alkyl; Y is CH₂; m is 0 or 1; n is the averagenumber of activated-PEG polymers conjugated to the protein; the —NH—group is an amine group of the protein; and N is a nitrogen of theprotein.

Another aspect is a nitroxylated protein having a conjugated PEG that isa maleimide-PEG, wherein the maleimide-PEG conjugated to an intrinsicthiol moiety of a cysteine residue or conjugated to a thiol moiety of athiolated lysine residue has the structure (VIII)

wherein Z represents the protein, S is the thiol group of the protein,R₃ is an alkylene or phenylene group, X is a terminal group, m is theaverage number of activated-PEG polymers conjugated to the protein, andn represents the average number of oxyethylene units of a PEG having anaverage molecular weight of about 2,000 to about 20,000 Daltons.

A further aspect is a nitroxylated protein having a conjugated PEG thatis a maleimide-PEG, wherein the maleimide-PEG conjugated to an intrinsicthiol moiety of a cysteine residue or conjugated to a thiol moiety of athiolated lysine residue has the structure (VIII)

wherein Z represents the protein, S is the thiol group of the protein,R₃ is an alkylene or phenylene group, X is a terminal group, m is theaverage number of activated-PEG polymers conjugated to the protein, andn represents the average number of oxyethylene units of a PEG having anaverage molecular weight of about 2,000 to about 20,000 Daltons.

Another aspect of the invention is a method for preparing a nitroxylatedprotein comprising reacting the protein with a nitroxylating agent offormula (IV):

wherein each of R₁, R₂, R₃, and R₄ is independently C₁-C₄ alkyl; Y isCH₂; and m is 0 or 1. method of claim G2, wherein each of R₁, R₂, R₃,and R₄ is independently —CH₃.

A further aspect is a method for making a nitroxylated protein using thenitroxylating agent of formula (II) or (IV) wherein the maleimide-PEGconjugated to an intrinsic thiol moiety of a cysteine residue has thestructure (VIII)

wherein Z represents the protein, R₃ is an alkylene or phenylene group,S is the thiol group of the protein, m is the average number ofactivated-PEG polymers conjugated to the protein, and n represents theaverage number of oxyethylene units of a PEG having an average molecularweight of about 2,000 to about 20,000 Daltons.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the results of ESI-TOF high accuracy mass spectroscopy of4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl (TEMPOL).

FIG. 2 shows the results of ESI-TOF high accuracy mass spectroscopyconfirming the molecular structure of 4-Succinimidyl-TEMPO-Carbonate(4-STC).

FIG. 3 shows thin layer chromatography (TLC) results performed onTEMPOL, N′-Disuccinimidyl carbonate (DSC), N-hydroxy-succinimide (NHS),reaction products from the reaction of TEMPOL and DSC at a 6 hourtimepoint, and the final 4-STC reaction product.

FIG. 4 shows Electron Paramagnetic Resonance (EPR) spectra for TEMPOLand PEGylated hemoglobin (MP4) both before and after polynitroxylation(PN-MP4).

FIG. 5 shows a size exclusion analysis profiles of PEGylated hemoglobin(PEG-Hb) and polynitroxylated PEGylated hemoglobin (PEG-Hb-PN).

FIG. 6 shows a characteristic UV-Vis spectrum for polynitroxylatedPEGylated hemoglobin (PEG-Hb-PN).

FIG. 7 shows UV-Vis spectra for plasma hemoglobin followingadministration of PEG-Hb-PN to rats.

FIG. 8 shows representative results of an experiment wherein PEGylatedhemoglobin was nitroxylated using 4-Succinimidyl-TEMPO-Carbonate (4-STC)at a molar excess of 1:5-1:100 over the PEGylated hemoglobin.

FIGS. 9 and 13 show MALDI-TOF spectra for human serum albumin (HSA).

FIGS. 10-12 and 14-16 show MALDI-TOF spectra for HSA polynitroxylatedusing 4-Succinimidyl-TEMPO-Carbonate at a molar excess of 1:5-1:100 overHSA.

FIG. 17 shows representative results of an experiment wherein HSA wasnitroxylated using 4-Succinimidyl-TEMPO-Carbonate (4-STC) at a molarexcess of 1:5-1:100 over HSA.

DEFINITIONS

When the terms “one,” “a” or “an” are used in this disclosure, they mean“at least one” or “one or more,” unless otherwise indicated.

“Activated polyalkylene oxide” or “activated PAO” as used herein referto a PAO molecule that has at least one functional group. A functionalgroup is a reactive moiety that interacts with free amines, sulfhydrylsor carboxyl groups on a molecule to be conjugated with PAO. For example,one such functional group that reacts with free sulfhydryls is amaleimide group. A functional group that reacts with free amines is asuccinimide group.

“Deoxyhemoglobin” or “unliganded hemoglobin” means any hemoglobin towhich no exogenous ligand is bound to heme.

“Hemoglobin” or “Hb” refers generally to a heme protein that transportsoxygen. In humans, each molecule of Hb has 4 subunits, 2 α-chainsubunits and 2 β-chain subunits, which are arranged in a tetramericstructure. Each subunit also contains one heme group, which is theiron-containing center that in the ferrous (Fe²⁺) binds the ligands O₂,NO or CO. Thus, each Hb molecule can bind up to 4 ligand molecules,making HbO₂, HbNO, or HbCO liganded compounds, respectively.Additionally, the hemoglobin may be liganded with mixtures of O₂, NO andCO.

“Hemoglobin based oxygen carriers” (HBOCs) refers to hemoglobins thatcarry oxygen, but are also useful for carrying other molecular gases,such as carbon monoxide and nitric oxide.

“High oxygen affinity” refers to hemoglobin that has been modified toexhibit an oxygen affinity greater than that of stroma free-hemoglobin(SFH). Thus, a “high oxygen affinity” Hb has a P50 less than that ofSFH, which has a P50 of 15 mmHg as measured at 37° C. and pH 7.4.

“Liganded hemoglobin” means hemoglobin to which an exogenous ligand isbound to heme. Common preferred ligands include oxygen, carbon monoxide,and nitric oxide.

“MalPEG” refers to maleimidyl polyethylene glycol, and includes amaleimidyl moiety attached to polyethylene glycol via a linker.

“MalPEG-Hb” refers to Hb to which maleimidyl-activated PEG has beenconjugated. The conjugation is performed by reacting MalPEG with thiolgroups (and to a lesser extent, amino groups) on the Hb to formMalPEG-Hb. Thiol groups are found in cysteine residues present in theamino acid sequence of Hb, such as the two intrinsic thiols at βCys 93,and can also be introduced by modifying surface amino groups to containa thiol group. An exemplary Ma1PEG-Hb known as MP4 (Sangart, Inc.) hasthe following formula:

wherein Hb is hemoglobin; S is a thiol group on the hemoglobin; n is thenumber of oxyethylene units of the 5,000-Dalton polyalkylene oxidepolymer; and m is the average number of maleimidyl-activatedpolyalkylene oxide polymers conjugated to the hemoglobin and is 7-8.

“Methemoglobin” or “metHb” refer to an oxidized form of Hb that containsiron in the ferric state. MetHb does not function as an oxygen or COcarrier. The term “methemoglobin %” as used herein refers to thepercentage of oxidized Hb to total Hb.

“Methoxy-PEG” or “mPEG-OH” refer to PEG wherein the hydrogen of thehydroxyl terminus is replaced with a methyl (—CH₃) group.

“Modified hemoglobin” or “modified Hb” refers to Hb that has beenaltered by a chemical reaction, such as intra- and inter-molecularcrosslinking, polymerization, conjugation, and/or recombinanttechniques, such that the Hb is no longer in its “native” state. As usedherein, the terms “hemoglobin” or “Hb” refer to both native unmodifiedHb and modified Hb, unless otherwise indicated.

“Nitrite reductase activity” or “NRA” is the ability of hemoglobin or ahemoglobin-based protein to reduce nitrite to nitric oxide. “Maximalnitrite reductase activity” is the maximum rate that hemoglobin or ahemoglobin-based protein is able to reduce nitrite to nitric oxide.“Initial nitrite reductase activity” is the initial rate that hemoglobinor a hemoglobin-based protein reduces nitrite to nitric oxide whennitrite is added to the fully deoxygenated protein.

The term “non-oxygenated” means that the heme protein or hemoglobin isin the non-liganded, deoxygenated state, or it is liganded with a gasother than O₂, such as NO or CO.

“Oxygen affinity” refers to the avidity with which an oxygen carrier,such as Hb, binds molecular oxygen. This characteristic is defined bythe oxygen equilibrium curve, which relates the degree of saturation ofHb molecules with oxygen (Y axis) with the partial pressure of oxygen (Xaxis). The position of this curve is denoted by the “P₅₀” value, whichis the partial pressure of oxygen at which the oxygen carrier ishalf-saturated with oxygen, and is inversely related to oxygen affinity.Hence, the lower the P₅₀, the higher the oxygen affinity. The oxygenaffinity of whole blood (and components of whole blood, such as redblood cells and Hb) can be measured by a variety of methods known in theart. (see, e.g., Winslow, R. M. et al., J. Biol. Chem. 1977,252:2331-37). Oxygen affinity may also be determined using acommercially available HEMOX™ Analyzer (TCS Scientific Corporation, NewHope, Pa.). (see, e.g., Vandegriff and Shrager in “Methods inEnzymology” (Everse et al., eds.) 232:460 (1994)) ; and Vandegriff, etal., Anal. Biochem. 256(1): 107-116 (1998)).

The term “oxygen therapeutic agent” as used herein refers to a hemeprotein that is capable of binding to and carrying molecular oxygen tocells/tissues/organs in need thereof. When administered in the form of aCO- or NO-liganded heme protein, once the CO or NO is released from theheme moiety, the heme groups are then free to bind to and carrymolecular oxygen.

“Polyethylene glycol” or “PEG” refer to a polymer of the generalchemical formula H(OCH₂CH₂)_(n) OH where “n” is greater than or equal to4, preferably about 45 to about 500, more preferably about 70 to about250, and most preferably about 90 to about 140, or about

115. The polymer can be substituted or unsubstituted, and the terminalhydroxy group can be replaced with a different conventional terminalgroup, such as methoxy or carboxy. PEGs are commercially available frommany sources (e.g., Carbowax™ (Dow Chemical, Midland, Mich.), Poly-G®(Arch Chemicals, Norwalk, Conn.) and Solbase).

“Polyethylene glycol-conjugated hemoglobin,” “PEG-Hb conjugate” or“PEG-Hb” refer to Hb to which at least one PEG is covalently attached.

“Solution” refers to a liquid mixture and the term “aqueous solution”refers to a solution that contains some water and may also contain oneor more other liquid substances with water to form a multi-componentsolution.

“Stroma-free hemoglobin” or “SFH” refer to Hb from which red blood cellmembranes have been removed.

“Surface-modified hemoglobin” refers to hemoglobin to which chemicalgroups, usually polymers, have been attached, such as dextran orpolyalkylene oxide. The term “surface-modified oxygenated hemoglobin”refers to Hb that is in the “R” state when it is surface modified.

“Terminal activity” is an indication of the percentage of PAO that isfunctionalized with a moiety capable of reacting with a reactive groupof the heme protein or hemoglobin. “100% Terminal activity” indicatesthat the molar excess of the PAO used in the conjugation reaction isexpressed on a basis that all of the PAO has a moiety capable ofreacting with a reactive group of the heme protein or hemoglobin. Forexample, if an available Mal-PEG has 80% terminal activity such that 80%of the PEGs are functionalized with Mal, and the Mal-PEG is used in20-fold molar excess over hemoglobin, then this molar ratio can beexpressed as a 16-fold molar excess of Mal-PEG over hemoglobin based on100% terminal activity.

“Thiolation” refers to a process that increases the number of sulfhydrylgroups on a molecule. For example, reacting a protein with2-iminothiolane (“2-IT”) converts free amines on the surface of theprotein to sulfhydryl groups. These sulfhydryl groups are then availablefor reaction with a thiol reactive moiety, such as a maleimide.

“Unliganded hemoglobin” refers to any hemoglobin containing at least oneheme moiety that is not liganded to a molecular gas such as oxygen,carbon monoxide or nitric oxide. As such, the hemoglobin is considered“unliganded” if only one of the heme moieties is not liganded to amolecular gas.

The term “heme protein” as used herein to any single or multiple chainprotein that bears a heme moiety that binds gases, such as oxygen,nitric oxide or carbon monoxide.

The term “nitroxide” as used herein refers to stable nitroxide freeradicals, their precursors and derivatives thereof. This term is not tobe confused with nitric oxide donor molecules.

DESCRIPTION OF THE INVENTION

The present invention generally relates to succinimide-activatednitroxyl compounds that can be used to nitroxylate proteins. Forexample, such nitroxyl compounds can be used to nitroxylate a hemeprotein such as hemoglobin. Nitroxylation of a heme protein with thesuccinimide-activated nitroxyl compounds counteracts the oxidation of NOand other biomolecules by oxidative substances, such as superoxide andhydrogen peroxide. The polynitroxylated heme proteins of the presentinvention are useful as (OTAs) that are capable of delivering molecularoxygen, carbon monoxide, nitric oxide, and mixtures thereof.

Succinimidvl Nitroxide Reagents

The present invention relates to succinimidyl nitroxide reagents thatcan be used to nitroxylate the amino groups of proteins. Thesuccinimidyl nitroxide reagents generally comprise a succinimide linkedto a nitroxide group, for example a TEMPO(2,2,6,6-tetramethyl-piperidine-1-oxyl) or PROXYL(2,2,5,5-tetramethylpyrrolidine-N-oxyl) nitroxide group. The linkagebetween the succinimide and the nitroxide can be, for example, a carboxylinkage or a carbonate linkage. Such reagents, for example nitroxylsuccinimidyl carbonates, are highly reactive, making coupling betweenthe amines of proteins and succinimide highly efficient.

For example, the present invention relates to a nitroxylating agent offormula (I):

wherein each of R₁, R₂, R₃, and R₄ is independently C₁-C₄ alkyl; X isoxygen, sulfur, nitrogen, phosphorus, or silicon; Y is CH₂; n is 0 or 1;and m is 0 or 1.

The nitroxylating agent of formula (I) can have a structure wherein X,Y, n, and m are as defined above and each of R₁, R₂, R₃, and R₄ is —CH₃.

Also, the nitroxylating agent of formula (I) can have a structurewherein R₁, R₂, R₃, R₄, Y, n, and m are as defined above and X is oxygenor sulfur.

Further, the nitroxylating agent of formula (I) can have a structurewherein R₁, R₂, R₃, R₄, Y, n, and m are as defined above and X isoxygen.

The nitroxylating agent of formula (I) can also have a structure whereinY, n, and m are as defined above, X is oxygen and each of R₁, R₂, R₃,and R₄ is —CH₃.

Additionally, the nitroxylating agent of formula (I) can have astructure wherein R₁, R₂, R₃, R₄, Y, and m are as defined above and n is0.

The nitroxylating agent of formula (I) can also have a structure whereinR₁, R₂, R₃, R₄, Y, and m are as defined above and n is 1.

Further, the nitroxylating agent of formula (I) can have a structurewherein R₁, R₂, R₃, R₄, Y, and n are as defined above and m is 0.

The nitroxylating agent of formula (I) can also have a structure whereinR₁, R₂, R₃, R₄, Y, and n are as defined above and m is 1.

For example, the nitroxylating agent of formula (I) can be selected from4-Succinimidyl-TEMPO-Carbonate (4-STC), 3-Succinimidyl-PROXYL-Carbonate(3-SPC), 4-succinimidyl-carboxy-TEMPO (4-SCT), and3-Succinimidyl-Carboxy-PROXYL (3-SCP). The structures of each of thesecompounds is shown below:

4-Succinimidyl-TEMPO-Carbonate (4-STC;1-(((2,2,6,6-tetramethyl-1-piperidinyloxy)-4-oxycarbonyl)oxy)-2,5-pyrrolidinedione)

3-Succinimidyl-PROXYL-Carbonate (3-SPC;1-(((2,2,5,5-tetramethyl-1-pyrrolidinyloxy)-3-oxycarbonyl)oxy)-2,5-pyrrolidinedione)

4-succinimidyl-carboxy-TEMPO (4-SCT;1-(((2,2,6,6-tetramethyl-1-piperidinyloxy)-4-carbonyl)oxy)-2,5-pyrrolidinedione)

3-Succinimidyl-Carboxy-PROXYL (3-SCP;1-(((2,2,5,5-tetramethyl-1-pyrrolidinyloxy)-3-carbonyl)oxy-2,5-pyrrolidinedione)

For example, the nitroxylating agent of formula (I) can have thefollowing structure:

Methods for the Synthesis of Succinimidyl Nitroxide Reagents

The succinimidyl nitroxide reagents can be synthesized using simpleone-step activation chemistry using readily available reagents.Furthermore, the activation reaction can be performed under mildconditions.

Thus, the invention further relates to a method for preparing anitroxylating agent of the formula (II):

comprising reacting a compound having the formula (III)

with N,N′-disuccinimidyl carbonate (DSC) in the presence of an organicbase; wherein each of R₁, R₂, R₃, and R₄ is independently C₁-C₄ alkyl; Xis oxygen, sulfur, nitrogen, phosphorus, or silicon; Y is CH₂; and m is0 or 1.

The method for preparing a nitroxylating agent of the formula (II) canhave structures of Formulae (II) and (III) wherein X, Y, and m are asdefined above and each of R₁, R₂, R₃, and R₄ is —CH₃.

The method for preparing a nitroxylating agent of the formula (II) canhave structures of Formulae (II) and (III) wherein R₁, R₂, R₃, and R₄,Y, and m are as defined above and X is oxygen or sulfur.

The method for preparing a nitroxylating agent of the formula (II) canhave structures of Formulae (II) and (III) wherein R₁, R₂, R₃, and R₄,Y, and m are as defined above and X is oxygen.

The method for preparing a nitroxylating agent of the formula (II) canhave structures of Formulae (II) and (III) wherein Y and m are asdefined above, X is oxygen and each of R₁, R₂, R₃, and R₄ is —CH₃.

The method for preparing a nitroxylating agent of the formula (II) canhave structures of Formulae (II) and (III) wherein R₁, R₂, R₃, and R₄,X, and Y are as defined above and m is 0.

The method for preparing a nitroxylating agent of the formula (II) canhave structures of Formulae (II) and (III) wherein R₁, R₂, R₃, and R₄,X, and Y are as defined above and m is 1.

The method for preparing a nitroxylating agent of the formula (II) canhave structures of Formulae (II) and (III) wherein R₁, R₂, R₃, and R₄,X, Y, and m are as defined above, and wherein the organic base comprisestriethylamine (TEA), N,N-diisopropylethylamine, 4-dimethylaminopyridine,pyridine, N-methylpiperidine, or a combination thereof.

The method for preparing a nitroxylating agent of the formula (II) canhave structures of Formulae (II) and (III) wherein R₁, R₂, R₃, and R₄,X, Y, and m are as defined above and wherein the organic base comprisestriethylamine.

The method for preparing a nitroxylating agent of the formula (II) canhave structures of Formulae (II) and (III) wherein R₁, R₂, R₃, and R₄,X, Y, and m are as defined above, wherein the organic base comprisestriethylamine, and wherein the compound of formula (III), theN,N′-disuccinimidyl carbonate, and the triethylamine are present in aratio of about 1:2:3.

In any of the above methods for preparing a nitroxylating agent of theformula (II), the reaction can be carried out at a temperature of about2° C. to about 30° C.; about 15° C. to about 25° C.; about 4° C.; orabout 20° C.

In any of the above methods for preparing a nitroxylating agent of theformula (II), the reaction can be allowed to proceed for about three toabout six hours.

In any of the above methods for preparing a nitroxylating agent of theformula (II), the reaction can be carried out in a polar aproticsolvent. The polar aprotic solvent can comprise acetonitrile (ACN),tetrahydrofuran (THF), ethyl acetate (EtOAc), acetone, dimethylformamide(DMF), dimethyl sulfoxide (DMSO), or a combination thereof. For example,the polar aprotic solvent can comprise acetonitrile.

A reaction scheme for the preparation of 4-Succinimidyl-TEMPO-Carbonateis shown below.

As shown above, 4-hydroxy-TEMPO (4-Hydroxy-2,2,6,6-tetramethylpiperidine1-oxyl) is used to prepare 4-succinmidyl-TEMPO-carbonate using one-stepactivation chemistry. Activation of TEMPOL to4-Succinimidyl-TEMPO-Carbonate is achieved by reaction of TEMPOL withN,N′-Disuccinimidyl-Carbonate in presence of triethylamine The organicbase (TEA in the reaction scheme above) is used as a catalyst thatdeprotonates the —OH group of the hydroxyl nitroxide (TEMPOL in thereaction scheme above), making it more reactive such that it can act asa nucleophile and attack the electrophilic carbonyl ofN,N′-Disuccinimidyl-Carbonate (DSC).

The invention is also directed to a method for preparing a nitroxylatingagent of the formula (IV):

comprising reacting a compound having the formula (V)

with N-hydroxysuccinimide (NHS) in the presence ofN,N′-dicyclohexylcarbodiimide (DCC); wherein each of R₁, R₂, R₃, and R₄is independently C₁-C₄ alkyl; Y is CH₂; and m is 0 or 1.

The method for preparing a nitroxylating agent of the formula (IV) canuse structures of formulae (IV) and (V) wherein Y and m are as definedabove in connection with formulae (IV) and (V) and wherein each of R₁,R₂, R₃, and R₄ is independently —CH₃.

In the method for preparing a nitroxylating agent of the formula (IV),the compound of formula (IV), the N-hydroxysuccinimide, and theN,N′-dicyclohexylcarbodiimide can be present in a reaction mixture in amolar ratio of about 1:1.1:1.1.

The method for preparing a nitroxylating agent of the formula (IV) canuse structures of formulae (IV) and (V) wherein Y and R₁, R₂, R₃, and R₄are as defined above in connection with formulae (IV) and (V) and m is0. The method can also use structures wherein m is 1.

In any of the above methods for preparing a nitroxylating agent of theformula (IV), the reaction is carried out at a temperature of about 2°C. to about 30° C.; about 15° C. to about 25° C.; about 4° C.; or about20° C.

In any of the above methods for preparing a nitroxylating agent of theformula (IV), the reaction can be allowed to proceed for about 6 toabout 24 hours.

In any of the above methods for preparing a nitroxylating agent of theformula (IV), the reaction can carried out at a pH of about 7.2 to about7.6; or at a pH of about 7.4.

Nitroxylated Proteins and Nitroxylated PAO-Modified Proteins

The present invention also relates to nitroxylated proteins which haveat least one nitroxylated amino group. The nitroxylated proteins arealso optionally conjugated to one or more a polyalkylene oxide (PAO)molecules, e.g., to one or more polyethylene glycol (PEG) molecules.

Polyethylene oxides for use in conjugating proteins include, but are notlimited to, polyethylene oxide, polypropylene oxide and apolyethylene/polypropylene oxide copolymer. The PAO has a molecularweight of about 2,000 to about 20,000 Daltons, preferably from about3,000 to about 10,000 Daltons, more preferably from 4,000 to about 6,000Daltons, and most preferably about 5,000 Daltons. The most common PAOpresently used to modify the surface of proteins is PEG because of itspharmaceutical acceptability and commercial availability. PEG isavailable in a variety of molecular weights based on the number ofrepeating subunits of ethylene oxide (i.e. —CH₂CH₂O—) within themolecule, to achieve a desired molecular weight based on the number andsize of the PEG molecules conjugated to a protein.

One or both of the terminal end groups of the PAO polymer are convertedinto a reactive functional group (“activated”). For example, PEG-OH hasbeen used to prepare PEG-halide, mesylate or tosylate, which is thenconverted to PEG-amine (“PEG-NH₂”) by performing a nucleophilicdisplacement reaction with aqueous ammonia (Zalipsky, S. et al., 1983,Eur. Polym. J. 19:1177-1183), sodium azide or potassium phthalimide. Theactivated PEG can then be conjugated to a protein through theinteraction of the PEG amine group (—“NH₂”) with a carboxyl group(“—COOH”) of the protein.

In addition to functionalizing PEG with an amine group and converting itto a maleimide group, PEGs that are activated therewith, are known to beused in the art. For example, PEG may be activated with p-nitrophenylcarbonate, aldehyde, aminopropyl, aminoethyl, thiol, aminoxy, hydrazide,and iodoacetamide, to name a few. Such functional PEG can be conjugatedto the surface amino acid side chains of proteins using known methods.

PEG-NH₂ can be further functionalized to conjugate with groups otherthan carboxyl. For example, U.S. Pat. No. 6,828,401 discloses thereaction of PEG-NH₂ with maleimide to form mPEG-maleimide. In thisreaction, mPEG-OH is reacted with a tosylating reagent(p-toluenesulfonyl chloride) and a base catalyst (triethyleneamine) inthe presence of an organic solvent (dichloromethane) to producemPEG-tosylate. The mPEG-tosylate is then reacted with 28% ammonia waterand maleic acid anhydride in an organic solvent mixture of N,N-dimethylacetamide (“DMAc”) and N-cyclohexylpyrrolidinone (“CHP”) toproduce a maleamic acid compound. This compound is then reacted withpentafluorophenyltrifluoroacetate in the presence of dichloromethane toproduce the mPEG-maleimide.

Alternatively, mPEG-maleimide can be made by reacting mPEG-OH with atosylating reagent (p-toluenesulfonyl chloride) and a base catalyst(triethyleneamine) in the presence of an organic solvent(dichloromethane) to produce mPEG-tosylate. The mPEG-tosylate is thenreacted with 28% ammonia to prepare mPEG-NH₂. The mPEG-NH₂ is thenreacted with N-methoxy carbonyl maleimide (MCM) in the presence ofsaturated sodium hydrocarbonate (NaHCO₃) to produce mPEG-maleimide.

Non-limiting examples of amino acid residue side chains of human Hb thatcan be modified using amine reactive chemistry for conjugation to PAOare presented in Table 1 below:

TABLE 1 Amine Reactive Chemistry and Potential Sites of ModificationResi- dues Positions Reacts With α-chain Lys 7, 11, 16, 40, 56,Succinimide; NPC (p-nitrophenyl carbonate); 60, 61, 90, 99, isocyanate;aldehyde; isothiocyanate; epoxides. 127 and 139 His 20, 45, 50, 58,Succinimide; NPC (p-nitrophenyl carbonate); 72, 87, 112 and isocyanate;aldehyde; isothiocyanate; epoxides. 122 Val 1 Succinimide; NPC(p-nitrophenyl carbonate); isocyanate; aldehyde; isothiocyanate;epoxides. β-chain Lys 8, 17, 59, 61, 65, Succinimide; NPC (p-nitrophenylcarbonate); 66, 82, 95, 120, isocyanate; aldehyde; isothiocyanate;epoxides. 132 and 144 His 2, 63, 77, 92, 97, Succinimide; NPC(p-nitrophenyl carbonate); 116, 117, 143 and isocyanate; aldehyde;isothiocyanate; epoxides. 146 Val 1 Succinimide; NPC (p-nitrophenylcarbonate); isocyanate; aldehyde; isothiocyanate; epoxides.

One method to increase the number of available conjugation sites on Hbis to introduce sulfhydryl groups (also known as thiolation), which tendto be more reactive with Ma1PEG than free amines. A variety of methodsare known for protein thiolation. In one method, protein free amines arereacted with succinimidyl 3-(2-pyridyldithio)propionate followed byreduction with dithiothreitol (“DTT”), or tris(2-carboxyethyl)phosphine(“TCEP”). This reaction releases the 2-pyridinethione chromophore, whichcan be used to determine the degree of thiolation. Amines can also beindirectly thiolated by reaction with succinimidylacetylthioacetate,followed by 50 mM hydroxylamine, or hydrazine at near-neutral pH.

Another method described in U.S. Pat. No. 5,585,484 maintains thepositive charge of the amino (α- or ε-) group of the Hb afterconjugation. This method involves amidination of the ε-amino groups ofHb by 2-IT to introduce sulfhydryl groups onto the protein. Thisapproach has at least two additional advantages over the previously usedsuccinimidyl chemistry: 1) the high reactivity and selectivity ofmaleimide groups with sulfhydryl groups facilitates the nearquantitative modification of the thiols, with a limited excess ofreagents and 2) the thiol group of 2-IT is latent and is generated onlyin situ as a consequence of the reaction of the reagent with the proteinamino groups. These advantages provide one additional benefit; theyallow simultaneous incubation of Hb with both the thiolating andPEGylation reagent for surface decoration.

For example, MalPEG can be conjugated to Hb by thiolating an amine ofthe Hb to introduce thiol groups on the surface of the Hb. The twointrinsic thiol groups of Hb that are available for reaction are atβCys93, and added thiol groups on the surface of the Hb can react withthe maleimide of the maleimidyl PAO to form a pegylated Hb conjugate.

The maleimide-PEG includes a linker to attach the maleimide to the PEG.Linkers can include, but are not limited to, alkylene such as ethylene,propylene, or isopropylene, phenylene, amide (—NH—C(O)—), or phenylcarbamate (e.g., -Ph-NH—C(O)—).

Non-limiting examples of amino acid residue side chains that can bemodified using thiol reactive chemistry are presented in Table 2 below:

TABLE 2 Thiol Reactive Chemistry and Potential Sites of ModificationResi- dues Positions Reacts With α-chain Cys 104 Maleimide;iodoacetamide; orthopyridyl- disulfide (OPSS); vinylsulfone. β-chain Cys93 and 112 Maleimide; iodoacetamide; orthopyridyl- disulfide (OPSS);vinylsulfone.

The molecular weight of the PAO-Hb can be regulated by the conjugationreaction. Conventional thought suggested that increasing the molarratios of the reactants would increase the number of PEG molecules boundto Hb. This included both the thiolation process of Hb (i.e. increasingthe molar ratio of thiolating agent to Hb) and the conjugation process(i.e. increasing the molar ratio of thiol activated PEG to thiolatedHb). However, these excess molar ratios resulted in the binding of only6±1 PEG molecules per Hb (see U.S. Pat. No. 7,501,499).

Recently it was determined that a greater number of PAO molecules couldbe bound to Hb using lower molar ratios of reactants. The number ofavailable thiol groups on Hb, before and after thiolation and afterconjugation, was determined using the dithiopyridine colorimetric assay(Ampulski, R. S. et al., 1969, Biochem. Biophys. Acta 32:163-169). HumanHb contains two intrinsic, reactive thiol groups at the β93cysteineresidues, which was confirmed by the dithiopyridine reaction. Afterthiolation of SFH with 2-IT, the number of reactive thiol groupsincreased from two to over seven. In this example, an average of 8 PEGmolecules was bound to Hb. This was achieved using a 7.5-fold molarexcess of 2-IT over SFH in the thiolation reaction and a 12-fold molarexcess of Ma1PEG over thiolated Hb in the conjugation reaction.

Hemoglobin is conjugated with polyalkylene oxide when it is in theoxygenated state to increase the oxygen affinity of the Hb-PAOconjugate.

Nitroxylated Proteins

The nitroxylated proteins have at least one nitroxylated amino group.The nitroxylated protein comprises at least one nitroxylated amino groupand the nitroxylated protein can have the structure (VI):

wherein Z represents the protein; each of R₁, R₂, R₃, and R₄ areindependently C₁-C₄ alkyl; X is oxygen, sulfur, nitrogen, phosphorus, orsilicon; Y is CH₂; m is 0 or 1; n is the average number of activated-PEGpolymers conjugated to the protein, the —NH— group is an amine group ofthe protein, and N is a nitrogen of the protein.

The nitroxylated protein can comprise at least one nitroxylated aminogroup, the nitroxylated protein can have the structure (VI) wherein R₁,R₂, R₃, R₄, Y, m, n, and Z are as defined above in connection withformula (VI) and X is oxygen or sulfur.

The nitroxylated protein can comprise at least one nitroxylated aminogroup, the nitroxylated protein can have the structure (VI) wherein R₁,R₂, R₃, R₄, Y, m, n, and Z are as defined above in connection withformula (VI) and X is oxygen.

The nitroxylated protein can comprise at least one nitroxylated aminogroup, the nitroxylated protein can have the structure (VI) wherein Y,m, n, and Z are as defined above in connection with formula (VI), X isoxygen, and each of R₁, R₂, R₃, and R₄ is —CH₃.

The nitroxylated protein can comprise at least one nitroxylated aminogroup, the nitroxylated protein having the structure (VII):

wherein Z represents the protein; each of R₁, R₂, R₃, and R₄ areindependently C₁-C₄ alkyl; Y is CH₂; m is 0 or 1; n is the averagenumber of activated-PEG polymers conjugated to the protein; the —NH—group is an amine group of the protein; and N is a nitrogen of theprotein.

The nitroxylated protein can comprise at least one nitroxylated aminogroup, the nitroxylated protein can have the structure (VI) or (VII)wherein X, Y, m, n, and Z are as defined in connection with formula (VI)or (VII) and each of R₁, R₂, R₃, and R₄ is —CH₃.

The nitroxylated protein can comprise at least one nitroxylated aminogroup, the nitroxylated protein having the structure (VI) or (VII)wherein R₁, R₂, R₃, R₄, X, Y, M, n, and Z are defined as in connectionwith formula (VI) or (VII) and m is 0.

The nitroxylated protein can comprise at least one nitroxylated aminogroup, the nitroxylated protein having the structure (VI) or (VII)wherein R₁, R₂, R₃, R₄, X, Y, M, n, and Z are defined as in connectionwith formulae (VI) or (VII) and wherein m is 1.

In any of the above nitroxylated proteins, the at least one nitroxylatedamino group can be the N-terminal amino group of the protein or anepsilon (ε)-amino group of a lysine residue.

The nitroxylated proteins can suitably be polynitroxylated proteins. Forexample, for nitroxylated proteins having structures of formulae (VI) or(VII), n is about 1 to about 25; n is at least about 2; n is at leastabout 5; n is at least about 10; or n is about 15 to about 20.

The polynitroxylated protein can be a polynitroxylated heme protein.

Heme proteins are useful in the practice of the present invention. Inaddition to tetrameric hemoglobin (Hb), this includes single chain(monomeric) natural or recombinant heme proteins, such as thosedescribed in BMC Structural Biology, 11:13, which is accessible athttp://www.biomedcentral.com/content/pdf/1472-6807-11-13.pdf. Otherexamples of heme proteins can be found in The Journal of ExperimentalBiology, 201: 1085-1098 (1998).

A variety of Hbs may be utilized with the present invention. The Hb maybe obtained from animal sources, such as human, bovine, porcine, orequine hemoglobin. Human Hb is preferred. The Hb can be obtained fromnatural sources or can be produced by known recombinant methods.

The hemoglobins of the present invention have a high oxygen affinitygreater than that of stroma-free hemoglobin. This means that thehemoglobins will have a P₅₀ less than 15 mmHg as measured at 37° C. andpH 7.4, preferably from about 2 to about 10 mmHg, and most preferablyfrom about 2 to about 8 mmHg or about 2 to about 5 mmHg.

For example, the nitroxylated protein can comprise a hemoglobinα-subunit, a hemoglobin β-subunit, a hemoglobin tetramer, or amyoglobin.

The nitroxylated protein can comprise a hemoglobin α-subunit, ahemoglobin β-subunit, a hemoglobin tetramer, a myoglobin, or an albumin.

Where the nitroxylated protein is an albumin, the nitroxylated proteincan comprise a serum albumin. The serum albumin can comprise human serumalbumin (HSA). HSA is a single polypeptide having 585 amino aicds. BSAcontains 60 lysines,17 pairs of disulfide bridges and one free cysteine,and has a molecular weight of approximately ˜67 ka

The nitroxylated protein can comprise a hemoglobin α-subunit or ahemoglobin β-subunit or hemoglobin tetramer. The nitroxylated proteincan comprise an animal hemoglobin α-subunit, an animal hemoglobinβ-subunit, or a hemoglobin tetramer comprising animal hemoglobinα-subunits and β-subunits.

The nitroxylated protein can comprise a human hemoglobin α-subunit, anhuman hemoglobin β-subunit, or a hemoglobin tetramer comprising humanhemoglobin α-subunits and β-subunits.

Where the nitroxylated protein is a hemoglobin tetramer, the hemoglobincan be intramolecularly cross-linked. Intramolecularly crosslinkedhemoglobins prevent dissociation into dimers and to avoid being clearedby the kidneys, extending circulation half-life. A variety of methodsare known in the art for intramolecularly crosslinking Hb. Chemicalcrosslinking reagents include glutaraldehyde (U.S. Pat. No. 7,005,414),polyaldehydes (U.S. Pat. No. 4,857,636), diaspirin (U.S. Pat. No.4,529,719), pyridoxyl-5′-phosphate (U.S. Pat. No. 4,529,719)trimesoyltris(methyl phosphate) (U.S. Pat. No. 5,250,665), dialkynes(for reaction with hemoglobin having an azide linker. See Foot et al.,Chem. Commun 2009, 7315-7317; Yang et al., Chem. Commun. 2010, 46:7557-7559) and hemoglobins can be crosslinked via recombinantmethodologies.

For example, the hemoglobin tetramer can comprise a cross-linked ααdimer or a cross-linked ββ dimer.

As shown in Table 1 above, the α- and β-subunits of human hemoglobinN-terminal valine residues that can be nitroxylated on the N-terminalamino group. In addition, the α- and β-subunits of human hemoglobincontain a number of lysine groups that can be nitroxylated at theε-amino group.

Additionally, the nitroxylated protein can comprise a human hemoglobinα-subunit. The human hemoglobin α-subunit can be nitroxylated at theα-amino group of the N-terminal valine residue. Further, the humanhemoglobin α-subunit can be nitroxylated at the ε-amino group of alysine residue selected from the group consisting of lysine-7,lysine-11, lysine-16, lysine-40, lysine-56, lysine-60, lysine-61,lysine-90, lysine-99, lysine-127, lysine-139, and a combination thereof.

The nitroxylated protein can also comprise a human hemoglobin β-subunit.The human hemoglobin β-subunit can be nitroxylated at the α-amino groupof the N-terminal valine residue. The human hemoglobin β-subunit canalso be nitroxylated at the ε-amino group of a lysine residue selectedfrom the group consisting of lysine-8, lysine-17, lysine-59, lysine-61,lysine-65, lysine-66, lysine-82, lysine-95, lysine-120, lysine-132,lysine-144, and a combination thereof.

Further, nitroxylated protein can comprise a hemoglobin tetramer and thehemoglobin tetramer can comprise about seventeen nitroxylated aminogroups.

Nitroxylated and PAO-Conjugated Proteins

The nitroxylated protein can also be conjugated to a polyalkylene oxide(PAO). The PAO can be a polyethylene glycol (PEG).

The PEG can have an average molecular weight of about 2,000 to about20,000 Daltons; about 3,000 to about 10,000 Daltons; about 4,000 toabout 6,000 Daltons; or about 5,000 Daltons.

The nitroxylated protein can also be conjugated to a PEG that is amaleimide-PEG. The maleimide can be linked to the PEG via an alkylene orphenylene linker. The alkylene linker can be an ethylene linker.

Also, the nitroxylated protein can have a conjugated PEG that is amaleimide-PEG conjugated to a thiol moiety of the protein selected fromthe group consisting of an intrinsic thiol moiety of a cysteine residueof the protein, a thiol moiety of a thiolated lysine residue of theprotein, and a combination thereof.

The nitroxylated protein can have a conjugated PEG that is amaleimide-PEG, wherein the maleimide-PEG conjugated to an intrinsicthiol moiety of a cysteine residue or conjugated to a thiol moiety of athiolated lysine residue has the structure (VIII)

wherein Z represents the protein, S is the thiol group of the protein,R₃ is an alkylene or phenylene group, X is a terminal group, m is theaverage number of activated-PEG polymers conjugated to the protein, andn represents the average number of oxyethylene units of a PEG having anaverage molecular weight of about 2,000 to about 20,000 Daltons.

The nitroxylated protein can have the structure of formula (VIII)wherein R₃ is ethylene.

The nitroxylated protein can have the structure of formula (VIII)wherein m is about 6 to about 10.

The nitroxylated protein can have the structure of formula (VIII)wherein X is methoxy (—OCH₃) or carboxylate (—COOH).

The nitroxylated protein can have the structure of formula (VIII)wherein the maleimide-PEG is conjugated to a thiol moiety of acysteine-93 residue of a hemoglobin β-subunit.

The nitroxylated protein having the structure of formula (VIII) whereinthe maleimide-PEG is conjugated to a thiol moiety of a thiolated lysineresidue of a hemoglobin α-subunit or β-subunit. The nitroxylated proteinhaving the structure of formula (VIII) wherein the thiolated lysineresidue is a thiolated lysine residue of a human hemoglobin α-subunitselected from the group consisting of lysine-7, lysine-11, lysine-16,lysine-40, lysine-56, lysine-60, lysine-61, lysine-90, lysine-99,lysine-127, lysine-139, and a combination thereof. The nitroxylatedprotein of formula (VIII) wherein the thiolated lysine residue is athiolated lysine residue of a human hemoglobin β-subunit selected fromthe group consisting of lysine-8, lysine-17, lysine-59, lysine-61,lysine-65, lysine-66, lysine-82, lysine-95, lysine-120, lysine-132,lysine-144, and a combination thereof.

Nitroxylated Hemoglobin Tetramers

A hemoglobin tetramer comprising at least one α-subunit or at least oneβ-subunit of any one of the nitroxylated hemoglobins described herein.

These hemoglobin tetramers can have at least one α-subunit and at leastone β-subunit of any one of the nitroxylated hemoglobins describedherein.

The hemoglobin tetramers can comprise two α-subunits and two β-subunitsof any one of the nitroxylated hemoglobins described herein.

The hemoglobin tetramers described herein wherein the hemoglobin isconjugated to on average 5 to 10 PAO molecules per tetramer, thehemoglobin is conjugated to on average 7.1 to 8.9 PAO molecules pertetramer.

The hemoglobin tetramer wherein the hemoglobin is oxygenated.

The hemoglobin tetramer wherein the hemoglobin is deoxygenated.

The hemoglobin tetramer wherein the hemoglobin is liganded with CO, NO,or a mixture of CO and NO.

The hemoglobin conjugates of the invention can be in oxygenated ordeoxygenated form, can be liganded to CO or NO, or can be a mixtureincluding two or more of these four forms. HbO₂ is prepared byequilibrating non-oxygenated hemoglobin with air, pure O₂ gas orO₂/nitrogen gas mixtures.

Deoxygenation can be performed by any method known in the art. Onesimple method is to expose the hemoglobin solution to an inert gas, suchas nitrogen, argon or helium. To assure that deoxygenation is relativelyhomogeneous, the Hb solution is circulated in this process. Monitoringdeoxygenation to attain desired levels may be performed by using aCo-oximeter 682 (Instrument Laboratories). If partial reoxygenation isdesired, deoxygenated Hb may be exposed to oxygen or to a gas mixturecontaining oxygen, such as air.

Gas exchange to replace molecular oxygen with another gas may beaccomplished through a gas-permeable membrane, such as a polypropyleneor cellulose acetate membrane. See, for example, published U.S. PatentApplication No. 2006/0234915. Commercially available gas-exchangedevices utilizing these membranes include the Celgard™ polypropylenemicroporous hollow fiber device from Hoechst-Celanese (Dallas, Tex.) orthe Cell-Pharm™ hollow fiber oxygenator from American Laboratory (EastLyme, Conn.). In the Hoechst-Celanese Celgard™ device, oxygenated Hb isdeoxygenated by passing an aqueous Hb solution through polypropylenemicroporous hollow filters at 10-100 ml/min/ft² while the system ispurged with nitrogen at 5-20 psi. The Hb is generally circulated forabout 5 to 30 minutes to achieve the desired percentage of deoxyHb.Another method for producing deoxygenated Hb comprises exposing a Hbsolution to a chemical reducing agent such as sodium ascorbate, sodiumdithionate and sodium bisulfite. Hb is partially deoxygenated byadjusting the reducing agent concentration, reaction time andtemperature. Alternatively, a reducing agent may be used tosubstantially deoxygenate Hb, and then oxygen may be reintroduced toform a partially deoxygenated product. For example, Hb can be exposed toa 100 mM concentration of sodium bisulfite for about one hour beforeadding antioxidants.

Hb can be liganded to CO using any known methods for formingoxyhemoglobin, simply by substituting CO for O₂. This generally involvesintroducing a source of CO to a solution of hemoglobin such that thehemoglobin becomes liganded with CO instead of O₂ (K. D. Vandegriff, etal., Biochem. J. 382:183-189 (2004)). Since hemoglobin has a higheraffinity for CO than it does for oxygen, it is not necessary to firstdeoxygenate the hemoglobin. Accordingly, the most convenient way offorming CO-Hb complexes is by introducing 100% gaseous CO to a solutionof hemoglobin.

HbNO can be prepared by reacting deoxygenated hemoglobin with nitricoxide gas, or by exposing CO-Hb to NO gas such that the NO exchanges forCO. HbNO can also be made by reacting deoxygenated hemoglobin with asmall NO-donor molecule like PROLI NONOate™ (i.e.,1-(hydroxy-NNO-azoxy)-L-proline, disodium salt; Cayman Chemical, AnnArbor, Mich.).

It should be noted that hemoglobin to which NO, a free radical, is boundto the amino acid side groups in the globin chain are not NO-Hbcomplexes as defined herein, since such compounds do not containdiatomic (nonionic) NO as a ligand in the heme pocket instead of oxygen.For example, nitrosylhemoglobin is formed when native hemoglobin isexposed to a NO donor under conditions that cause it to bind to freesulfhydryl groups (U.S. Pat. No. 6,627,738). Such nitrosylhemoglobinsstill carry oxygen, whereas the NO-Hb complexes of the present inventiondo not. Furthermore, when the modified hemoglobin is formed by areaction directed towards sulfhydryl moieties such as described above,these moieties are no longer available for NO binding.

Methods for the Nitroxylation of Proteins and PAO-Modified Proteins

The present invention also relates to methods for nitroxylation ofproteins, including PAO-modified proteins. The methods of the presentinvention provide site-specific nitroxylation and can be performed underfavorable reaction conditions. Nitroxylated-PEGylated hemoglobinproduced using these methods has enhanced circulation time and proteinstability, as well as a high oxygen affinity.

The nitroxylated protein can be prepared by reacting the protein with anitroxylating agent of formula (II)

wherein each of R₁, R₂, R₃, and R₄ is independently C₁-C₄ alkyl; X isoxygen, sulfur, nitrogen, phosphorus, or silicon; Y is CH₂; and m is 0or 1.

The method of making a nitroxylated protein wherein the nitroxylatingagent of formula (II) has R₁, R₂, R₃, R₄, Y, and m as defined inconnection with formula (II) and X is oxygen or sulfur.

The method of making a nitroxylated protein wherein the nitroxylatingagent of formula (II) has R₁, R₂, R₃, R₄, Y, and m as defined inconnection with formula (II) and X is oxygen.

The method of making a nitroxylated protein wherein the nitroxylatingagent of formula (II) has Y and m as defined in connection with formula(II) X is oxygen and each of R₁, R₂, R₃, and R₄ is —CH₃.

A method for preparing a nitroxylated protein comprising reacting theprotein with a nitroxylating agent of formula (IV):

wherein each of R₁, R₂, R₃, and R₄ is independently C₁-C₄ alkyl; Y isCH₂; and m is 0 or 1. method of claim G2, wherein each of R₁, R₂, R₃,and R₄ is independently —CH₃.

The method of making a nitroxylated protein wherein the nitroxylatingagent of formula (II) or (IV) has X, Y, and m as defined in connectionwith formula (II) or (IV) and each of R₁, R₂, R₃, and R₄ is —CH₃.

The method of making a nitroxylated protein wherein the nitroxylatingagent of formula (II) or (IV) has R₁, R₂, R₃, R₄, X, and Y as defined inconnection with formula (II) or (IV) and m is 0.

The method of making a nitroxylated protein wherein the nitroxylatingagent of formula (II) or (IV) has R₁, R₂, R₃, R₄, X, and Y as defined inconnection with formula (II) or (IV) and m is 1.

The method for making a nitroxylated protein wherein the ratio of thenitroxylating agent of formula (II) or (IV) is present at about a 5- toabout 100-fold molar excess over the protein.

The method for making a nitroxylated protein using the nitroxylatingagent of formula (II) or (IV) wherein the protein comprises an α- orβ-subunit of a hemoglobin tetramer. The method where the proteincomprises a hemoglobin tetramer. The method wherein the hemoglobintetramer is a non-oxygenated hemoglobin tetramer. The method wherein thenon-oxygenated hemoglobin tetramer is a CO-liganded hemoglobin tetramer.The method wherein the hemoglobin tetramer is a deoxygenated hemoglobintetramer.

The method for making a nitroxylated protein using the nitroxylatingagent of formula (II) or (IV) wherein the reaction is carried out at atemperature of about 2° C. to about 30° C.; about 15° C. to about 25°C.; about 2° C. to about 8° C.; about 4° C.; or about 20° C.

The method for making a nitroxylated protein using the nitroxylatingagent of formula (II) or (IV) wherein the reaction is allowed to proceedfor about three to about 20 hours; about three to about six hours; orabout 16 hours.

The method for making a nitroxylated protein using the nitroxylatingagent of formula (II) or (IV) wherein the reaction is carried out in anaqueous solvent.

The method for making a nitroxylated protein using the nitroxylatingagent of formula (II) or (IV) wherein the reaction is carried out at apH of about 6.5 to about 8.5; a pH of about 7.5; or a pH of about 7.2.

The method for making a nitroxylated protein using the nitroxylatingagent of formula (II) or (IV) wherein the protein comprises a hemoglobintetramer, the reaction is carried out at a pH of about 7.2 and atemperature of about 2° C. to about 8° C. and is allowed to proceed forabout sixteen hours, and wherein the method yields a nitroxylatedhemoglobin tetramer having about 17 nitroxylated amino groups.

The method for making a nitroxylated protein using the nitroxylatingagent of formula (II) or (IV) wherein the protein comprises a hemoglobintetramer, and the nitroxylating agent is present at about a 10- to about100-fold molar excess over the hemoglobin tetramer.

The method for making a nitroxylated protein of using the nitroxylatingagent formula (II) or (IV) wherein the product of the reaction is anitroxylated protein of formula (VI) or (VII) or a hemoglobin tetramersdescribed herein.

The method for making a nitroxylated protein using the nitroxylatingagent of formula (II) or (IV) further comprising conjugating the proteinto a polyalkylene oxide (PAO).

The method for making a nitroxylated protein using the nitroxylatingagent of formula (II) or (IV) further comprising adding succinimidylvalerate PAO to the protein in the aqueous diluent to form aPAO-valerate conjugated protein. The method further comprising mixingthe protein with 2-iminothiolane (2-IT) in an aqueous diluent to form athiolated protein; and adding PAO-maleimide to the thiolated protein inthe aqueous diluent to form a PAO-maleimide conjugated protein. Themethod wherein the PAO is a polyethylene glycol (PEG).

The method for making a nitroxylated protein using the nitroxylatingagent of formula (II) or (IV) further comprising conjugating the proteinto a polyalkylene oxide (PAO) and wherein the PAO is a polyethyleneglycol (PEG) wherein the PEG has an average molecular weight of about2,000 to about 20,000 Daltons; about 3,000 to about 10,000 Daltons;about 2,000 to about 6,000 Daltons; or about 5,000 Daltons.

The method for making a nitroxylated protein using the nitroxylatingagent of formula (II) or (IV) wherein the PEG is a maleimide-PEG. Themethod wherein the maleimide is linked to the PEG via an alkylene orphenylene linker. The method wherein the alkylene linker is an ethylenelinker. The method wherein the maleimide-PEG is conjugated to a thiolmoiety of the protein selected from the group consisting of an intrinsicthiol moiety of a cysteine residue of the protein, a thiol moiety of athiolated lysine residue of the protein, and a combination thereof.

The method for making a nitroxylated protein using the nitroxylatingagent of formula (II) or (IV) wherein the maleimide-PEG conjugated to anintrinsic thiol moiety of a cysteine residue has the structure (VIII)

wherein Z represents the protein, R₃ is an alkylene or phenylene group,S is the thiol group of the protein, m is the average number ofactivated-PEG polymers conjugated to the protein, and n represents theaverage number of oxyethylene units of a PEG having an average molecularweight of about 2,000 to about 20,000 Daltons.

The method wherein the maleimide-PEG conjugated to an intrinsic thiolmoiety of a cysteine residue has the structure (VIII) wherein R₃ isethylene. The method wherein the protein comprises a hemoglobinα-subunit, a hemoglobin β-subunit, a hemoglobin tetramer, a myoglobin,or an albumin. The method wherein the protein comprises a serum albumin.The method wherein the serum albumin comprises human serum albumin(HSA). The method wherein the protein comprises a hemoglobin tetramer.

The method wherein the hemoglobin tetramer comprises a cross-linked ααdimer or a cross-linked ββ dimer. The method wherein the maleimide-PEGis conjugated to a thiol moiety of a cysteine-93 residue of a hemoglobinβ-subunit. The method wherein the maleimide-PEG is conjugated to a thiolmoiety of a thiolated lysine residue of a hemoglobin α-subunit orβ-subunit. The method wherein the thiolated lysine residue is athiolated lysine residue of a human hemoglobin α-subunit selected fromthe group consisting of lysine-7, lysine-11, lysine-16, lysine-40,lysine-56, lysine-60, lysine-61, lysine-90, lysine-99, lysine-127,lysine-139, and a combination thereof. The method wherein the thiolatedlysine residue is a thiolated lysine residue of a human hemoglobinβ-subunit selected from the group consisting of lysine-8, lysine-17,lysine-59, lysine-61, lysine-65, lysine-66, lysine-82, lysine-95,lysine-120, lysine-132, lysine-144, and a combination thereof.

The method for making a nitroxylated protein using the nitroxylatingagent of formula (II) or (IV) wherein the 2-iminithiolane is present ata concentration of between about 7- and about 15-fold molar excess overthe protein concentration; between about 7- and about 8-fold molarexcess over the protein concentration; or at about 7-0.5-fold molarexcess over the protein concentration.

The method for making a nitroxylated protein using the nitroxylatingagent of formula (II) or (IV) wherein the PAO-maleimide is present at aconcentration of between about 9- and about 20-fold molar excess overthe protein concentration; between about 9- and about 15-fold molarexcess over the protein concentration; or about a 12-fold molar excessover the protein concentration.

The method for making a nitroxylated protein using the nitroxylatingagent of formula (II) or (IV) wherein the thiolation step is carried outat a pH of between about 7 and about 9; or a pH of about 8.5.

The method for making a nitroxylated protein using the nitroxylatingagent of formula (II) or (IV) wherein the step of adding thePAO-maleimide to the thiolated protein to form a PAO-maleimideconjugated protein is carried out at a pH of between about 6.5 and about8.5; or at a pH of about 7.5.

The method for making a nitroxylated protein using the nitroxylatingagent of formula (II) or (IV) wherein the PEG is an SVA-PEG. The methodwherein the succinimide is linked to the PEG via —C(O)—(CH₂)₄—. TheSVA-PEG can be conjugated to an amino moiety of the protein selectedfrom an ε-amino moiety of a lysine residue of the protein, an α-aminomoiety of a terminal valine residue of the protein, or a combinationthereof. The SVA-PEG can also be conjugated to an ε-amino moiety of alysine residue of the protein or an α-amino moiety of a terminal valineresidue of the protein has the structure (IX)

wherein Z is the protein, N is a nitrogen of the protein, X is aterminal group, m is the number of activated-PEG polymers conjugated tothe protein, and n is the average number of oxyethylene units of a PEGhaving an average molecular weight of from about 2,000 to about 20,000Daltons.

X is a terminal group of the PAO, and can be hydroxy, aryloxy such asbenzyloxy, or C₁-C₂₀ alkoxy, more preferably C₁-C₁₀ alkoxy group, andstill more preferably a C₁-C₅ alkoxy group such as methoxy or ethoxy.

The method for making a nitroxylated protein using the nitroxylatingagent of formula (II) or (IV) wherein for formula IX m is on averagefrom about 6 to about 10 PAO molecules per tetramer. The SVA-PEG isconjugated to an ε-amino moiety of a lysine residue of a hemoglobinα-subunit or β-subunit. The SVA-PEG is conjugated to an α-amino moietyof a terminal valine residue of a hemoglobin α-subunit or β-subunit.

The method for making a nitroxylated protein using the nitroxylatingagent of formula (II) or (IV) wherein for formula IX the lysine residueis a lysine residue of a human hemoglobin α-subunit selected from thegroup consisting of lysine-7, lysine-11, lysine-16, lysine-40,lysine-56, lysine-60, lysine-61, lysine-90, lysine-99, lysine-127,lysine-139, and a combination thereof. The lysine residue is a lysineresidue of a human hemoglobin β-subunit selected from the groupconsisting of lysine-8, lysine-17, lysine-59, lysine-61, lysine-65,lysine-66, lysine-82, lysine-95, lysine-120, lysine-132, lysine-144, anda combination thereof.

The method for making a nitroxylated protein using the nitroxylatingagent of formula (II) or (IV) wherein the protein is conjugated to PAOprior to nitroxylation of the protein. The step of adding thePAO-maleimide to the thiolated protein to form a PAO-maleimideconjugated protein can be performed concurrently with nitroxylation ofthe protein. The step of adding the succinimidyl valerate PAO to theprotein to form a PAO-valerate conjugated protein is performedconcurrently with nitroxylation of the protein.

In any of the above methods, the degree of nitroxyl substitution can beevaluated and quantified using electron paramagnetic resonance (EPR) orMALDI-TOF mass spectroscopy

Pharmaceutical Compositions

The PAO-Hb conjugates of the present invention can be formulated as apharmaceutical composition comprising the PAO-Hb conjugate in apharmaceutically acceptable carrier for parenteral administration, suchas an aqueous diluent. The concentration of the PAO-Hb conjugate in thecarrier can vary according to the application. Preferably, the PAO-Hbconjugate concentration ranges from about 0.1 g/dl to about 10 g/dl,more preferably from about 2.0 g/dl to about 8.0 g/dl, and mostpreferably about 4.0 to about 6.0 g/dl. The selection of an appropriateconcentration of hemoglobin depends on the colloidal osmotic (oncotic)properties of the final hemoglobin product. Preferably, the compositionsof the invention are normo-oncotic as compared to whole blood orhyperoncotic as compared to plasma. The hemoglobin concentration can beadjusted to obtain the desired oncotic pressure for each indication.

When the composition is formulated as a parenteral, the solutiongenerally comprises a physiologically compatible electrolyte carrierisosmotic with whole blood and which maintains the reversible oxygen-,CO- or NO-carrying and delivery properties of the hemoglobin.

The pharmaceutically acceptable carrier can be an aqueous diluent. Theaqueous diluent can comprise an aqueous solution of a colloid or anaqueous solution of a non-oxygen carrying component, such as an aqueoussolution of proteins such as albumin, an aqueous solution ofglycoproteins, an aqueous solution of polysaccharides, or a combinationthereof. The aqueous diluent can comprise an aqueous cell-free solution.

Suitable aqueous diluents include, but are not limited to, physiologicalsaline, a saline-glucose mixture, Ringer's solution, lactated Ringer'ssolution, Locke-Ringer's solution, Krebs-Ringer's solution, Hartmann'sbalanced saline, heparinized sodium citrate-citric acid-dextrosesolution, an acetate solution, a multiple electrolyte solution (e.g.,Plasma Lyte® or Plasma Lyte-A® from Baxter International, Deerfield,Ill.), a lactobionate solution, and polymeric plasma substitutes, suchas polyethylene oxide, polyvinyl pyrrolidone, polyvinyl alcohol, anethylene oxide-propylene glycol condensate, or a combination thereof.

The composition can additionally comprise pharmaceutically-acceptablefillers, salts, and other materials well-known in the art, the selectionof which depends on the dosage form, the condition being treated, theparticular purpose to be achieved according to the determination of theordinarily skilled artisan in the field and the properties of suchadditives. For example, the composition can include physiologicalbuffers, carbohydrates (e.g. glucose, mannitol, or sorbitol), alcoholsor poly alcohols, pharmaceutically acceptable salts (e.g., sodium orpotassium chloride), surfactants (e.g., polysorbate 80), anti-oxidants,anti-bacterial agents, oncotic pressure agents (e.g. albumin orpolyethylene glycols) or reducing agents (e.g., ascorbic acid,glutathione, or N-acetyl cysteine).

The pharmaceutical compositions have a viscosity of at least about 2centipoise (cP). More specifically, the viscosity ranges from about 2 toabout 5 cP, and particularly about 2.5 to about 4.5 cP.

In order to avoid complications in administration, the pharmaceuticalcomposition is of high purity, i.e. free from stroma, phospholipids, andpyrogens, having an endotoxin level of no more than 0.25 EU/ml, asmeasured by the LAL (limulus amebocyte lysate) test, and having lessthan 8% methemoglobin.

Pharmaceutical compositions can be administered parenterally, such as bysubcutaneous, intravenous, or intramuscular injection, or as largevolume parenteral solutions. The compositions can also be administeredby gavage.

A typical dose of hemoglobin conjugate as a therapeutic agent can befrom about 1 to about 15,000 milligrams of hemoglobin per kilogram ofpatient body weight. For example, when used as an oxygen therapeutic,the dosage will range between 100 to 7500 mg/kg patient body weight,more preferably 500 to 5000 mg/kg body weight, and most preferably 700to 3000 mg/kg body weight. Thus, a typical dose for a human patientmight be from a gram to over 1000 grams. It will be appreciated that theunit content of active ingredients contained in an individual dose ofeach dosage form need not in itself constitute an effective amount, asthe necessary effective amount could be reached by administration of anumber of individual doses. The selection of dosage depends upon thedosage form utilized, the condition being treated, and the particularpurpose to be achieved according to the determination of those skilledin the art.

Methods of Treatment

The PAO-Hb conjugates and pharmaceutical compositions can be used todeliver oxygen, CO and/or NO to a subject. A method of deliveringoxygen, nitric oxide, carbon monoxide or mixtures thereof to tissue andreducing nitrite to produce further endogenous nitric oxide (NO) in themicrovasculature includes administering the hemoglobin conjugate or thecomposition to a subject in need thereof, wherein followingadministration, the hemoglobin becomes unliganded and converts nitriteto nitric oxide in the microvasculature.

The hemoglobin conjugates and compositions thereof of the invention canbe used: to treat acute liver failure, beta thalassemia, a burn, chroniccritical limb ischemia, carbon dioxide or cyanide poisoning, chronicobstructive pulmonary disease (COPD) (e.g., acute exacerbations),congestive heart failure (e.g., acute heart failure, chronic heartfailure), hypoxia (e.g., high altitude use including for pulmonaryedema, decompression sickness), malaria (e.g., cerebral malaria(Falciparum occlusive events), organ ischemia (e.g., acute bowelischemia (torsion), acute bowel ischemia (embolism), cardiogenic shock,acute vascular organ ischemia, stroke (before CAT scan), stroke (afterCAT scan), myocardial infarction/severe cardiac ischemia), peripheralvascular disease, porphyria, pre-eclampsia in pregnancy, sepsis, sicklecell disease (e.g., stroke/transient ischemic attack, splenicsequestration, hepatic sequestration, priapism), retinaldisease/intra-ocular condition (e.g., central retinal artery occlusion,central venous occlusion), testicular torsion, trauma/shock (e.g.,traumatic hemorrhagic shock, non-traumatic hemorrhagic shock,pre-hospital/field use (military/emergency), traumatic braininjury/blast), ulcers, or vasospasm; as an adjunct to angioplasty, as anadjunct for plastic surgery (skin flaps) (e.g., acute treatment, chronictreatment), or as an adjunct in implanting a ventricular assist device;as a blood substitute (e.g., for acute blood loss, Jehovah's Witness,difficult to cross-match patient, rare blood group, sickle aplasticcrisis, sickle cell anemia perioperative management, acute hemolyticanemia (autoimmune), acute hemolytic anemia (toxin), or other refractoryanemia), a cardioprotectant, a cryopreservative, a hemodialysis adjunct,an oncology agent (e.g., adjunct to radiotherapy or chemotherapy, solidtumors), an organ preservative (e.g., ex vivo, in donor, in recipient),a performance enhancement agent (e.g., civilian/athletic, military), asurgery adjunct (e.g., cardiopulmonary bypass (prime), cardiopulmonarybypass (adjustment), lung ischemia, pre-surgery conditioning, rupturedaortic aneurysm, replacement of thoracic aorta (dissection oraneurysm)), or a wound healing agent; in imaging (x-ray or magneticresonance imaging (MRI)); to improve lung function (e.g., acute lunginjury, chronic lung injury, transient viral pneumonia, neonataldistress syndrome); or a combination thereof. Such uses includeadministration of the conjugate or composition to a subject in needthereof.

Further, the hemoglobins and compositions of the invention can be usedto treat non-traumatic hemorrhagic shock, pre-hospital setting trauma,traumatic hemorrhagic shock, acute lung injury, adult respiratorydistress syndrome, traumatic brain injury, stroke, solid tumor cancer,organ degradation (ex-vivo), organ degradation (in recipient), severesepsis/septic shock, myocardial infarction/cardiac ischemia, cardiogenicshock, acute heart failure, pulmonary embolism, various conditions bysurgery (e.g., adjunct to angioplasty, adjunct to thoracic aorticrepairs, adjunct to cardiopulmonary bypass, priming solution forcardiopulmonary bypass), or a combination thereof.

The numerous clinical settings in which the hemoglobins and compositionsof the present invention are useful include the following:

Trauma. An acute loss of whole blood can result in a fluid shift fromthe interstitial and intracellular spaces to replace the lost volume ofblood while shunting of blood away from the low priority organsincluding the skin and gut. Shunting of blood away from organs reducesand sometimes eliminates O₂ levels in these organs and results inprogressive tissue death. The primary goal is to oxygenate affectedtissues. This trauma can be in a pre-hospital setting or can result intraumatic hemorrhagic shock or traumatic brain injury.

Ischemia. The conjugates and compositions thereof can also be used todeliver oxygen, CO, and/or NO to areas that red blood cells or manyother oxygen therapeutics cannot penetrate. These areas can include anytissue areas that are located downstream of obstructions to red bloodcell flow, such as areas downstream of thrombi, sickle cell occlusions,arterial occlusions, angioplasty balloons, surgical instrumentation, andany tissues that are suffering from oxygen starvation or are hypoxic.All types of tissue ischemia can be treated including, for example,stroke, emerging stroke, transient ischemic attacks, myocardial stunningand hibernation, acute or unstable angina, emerging angina, infarct, andthe like. In particular, conditions resulting in ischemia include acuteheart failure, cardiogenic shock, myocardial infarction/cardiacischemia, stroke, pulmonary embolism, non-traumatic hemorrhagic shock,or cerebrovascular trauma.

Hemodilution. In this application, the therapeutic is administered toreplace (or substitute for) the O₂ levels of the removed autologousblood. This permits the use of the removed autologous blood fornecessary transfusions during and after surgery. One such surgeryrequiring pre-operative blood removal would be a cardiopulmonary bypassprocedure.

Sepsis/Septic Shock. In sepsis, some patients may become hypertensive inspite of massive fluid therapy and treatment with vasoconstrictoragents. In this instance, the overproduction of nitric oxide (NO)results in lowered blood pressure. Therefore hemoglobin is a desirableagent for treatment of these patients because hemoglobin binds NO with ahigh avidity.

Hypoxemia. When a patient has acute lung injury caused by eitherpneumonia or pancreatitis, hypoxemia can be observed and can bealleviated by providing the hemoglobins or compositions of the inventionto oxygenate the affected tissues.

Cancer. Delivery of O₂ to the hypoxic inner core of a solid tumor massincreases its sensitivity to radiotherapy and chemotherapy. Because themicrovasculature of a tumor is unlike that of other tissues,sensitization through increasing O₂ levels requires O₂ be unloadedwithin the hypoxic core. In other words, the P50 should be very low toprevent early unloading of the O₂, increasing the O₂ levels, to insureoptimal sensitization of the tumor to subsequent radiation andchemotherapy treatments.

Surgery. The hemoglobins and compositions of the invention can be usedduring various surgical procedures. For example, they can be used as anadjunct to angioplasty, thoracic aortic repairs, during acardiopulmonary bypass procedure or as a cardiopulmonary primingsolution.

Organ Perfusion. During the time an organ is maintained ex vivo or in anorgan donation recipient, maintaining O₂ content helps preservestructural and cellular integrity and minimizes infarct formation. Thehemoglobins and compositions can sustain the oxygen requirements forsuch an organ.

The hemoglobins and compositions thereof can also be used in non-humans,such as domestic animals (e.g., livestock and companion animals such asdogs, cats, horses, birds, reptiles. It is contemplated that the presentinvention finds utility in the emergency treatment of domestic and wildanimals suffering a loss of blood due to injury, hemolytic anemias, etc.Veterinary uses include treatment of loss of blood due to injury,hemolytic anemia, equine infectious anemia, feline infectious anemia,bacterial infection, Factor IV fragmentation, hypersplenation andsplenomegaly, hemorrhagic syndrome in poultry, hypoplastic anemia,aplastic anemia, idiopathic immune hemolytic conditions, irondeficiency, isoimmune hemolytic anemia, microangiopathic hemolyticanemia, parasitism, or surgical-anesthesia induced brain damage.

EXAMPLES Example 1 Synthesis of 4-Succinimidyl-TEMPO-Carbonate (4-STC;1-(((2,2,6,6-tetramethyl-1-piperidinyloxy)-4-oxycarbonyl)oxy)-2,5-pyrrolidinedione)

One gram of 4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl (TEMPOL) wasdissolved in 20 mL anhydrous acetonitrile and mixed for five minutes atroom temperature. Once the TEMPOL dissolved, 2.975 g ofN,N′-Disuccinimidyl carbonate (DSC) (2 eq) and 2.425 mL of triethylamine(3.0 eq) were added to the reaction. The reaction was performed at roomtemperature, under anaerobic condition for 6-8 hours. After reactioncompletion, the solvent was evaporated under reduced pressure. Theresidue was dissolved in ethyl acetate and washed with saturated aqueousCuSO₄ solution. The organic phase was separated and 0.5 g of Na₂SO₄ pergram of TEMPOL was added to the organic phase. The solution was mixedfor 15 min at room temperature followed by filtration. Filtered solutionwas evaporated under reduced pressure. The product,4-Succinimidyl-TEMPO-Carbonate, was precipitated by adding n-heptane,filtered, and dried under vacuum at room temperature.

A reaction scheme for the preparation of 4-Succinimidyl-TEMPO-Carbonateis shown below:

Example 2 Analysis of 4-Succinimidyl-TEMPO-Carbonate (4-STC)

ESI-TOF high accuracy mass spectroscopy was performed on the startingmaterial TEMPOL and the final product 4-Succinimidyl-TEMPO-Carbonate(4-STC) to confirm conversion of TEMPOL to 4-STC (FIGS. 1 and 2).

In addition, as shown in FIG. 3 thin layer chromatography was performedon the starting materials TEMPOL (lane 1) and N,N′-Disuccinimidylcarbonate (DSC, lane 3), and the reaction product at 6 hours (lane 4)and the final product 4-Succinimidyl-TEMPO-Carbonate after precipitation(lane 5). Lane 2 was loaded with N-hydroxy-succinimide (NHS), a reactionby-product that is released from DSC during the reaction. As can be seenfrom FIG. 3, the final product does not contain any of the startingmaterials or by-products.

Example 3 Synthesis of 3-Succinimidyl-PROXYL-Carbonate (3-SPC;1-(((2,2,5,5-tetramethyl-1-pyrrolidinyloxy)-3-oxycarbonyl)oxy)-2,5-pyrrolidinedione)

3-Succinimidyl-PROXYL-Carbonate can be prepared using a method similarto those described above in Example 1 for4-Succinimidyl-TEMPO-Carbonate, using3-hydroxy-2,2,5,5-tetramethylpyrrolidin-1-oxyl instead of TEMPOL as astarting material.

Example 4 Synthesis of 4-succinimidyl-carboxy-TEMPO (4-SCT;1-4(2,2,6,6-tetramethyl-1-piperidinyloxy)-4-carbonyl)oxy)-2,5-pyrrolidinedione)

One gram of 4-Carboxy-2,2,6,6-tetramethylpiperidin-1-oxyl (4-CarboxyTEMPO) was dissolved in 75 mL tetrahydrofuran and mixed for five minutesat room temperature. Once the 4-Carboxy TEMPO dissolved, 0.632 g ofN-Hydroxysuccinimide (NHS) (1.1 eq) and 1.15 g ofN,N′-Dicyclohexylcarbodiimide (1.1 eq) were added to the reaction. Thereaction was performed at room temperature under anaerobic conditionsfor 24 hours. After reaction completion, the solution was filtered andevaporated under reduced pressure. The residue was dissolved in ethylacetate and washed with water. The organic phase was separated and 0.5 gof Na₂SO₄ per gram of 4-Carboxy TEMPO was added to the organic phase.The solution was mixed for 15 min at room temperature followed byfiltration. Filtered solution was evaporated under the reduced pressure.The product, 4-Succinimidyl-Carboxy-TEMPO was precipitated by addingn-Heptane, filtered, and dried under vacuum at room temperature.

A reaction scheme for the preparation of 4-Succinimidyl-Carboxy-TEMPO isshown below:

Example 5 Synthesis of 3-Succinimidyl-Carboxy-PROXYL (3-SCP;1-(((2,2,5,5-tetramethyl-1-pyrrolidinyloxy)-3-carbonyl)oxy-2,5-pyrrolidinedione)

3-Succinimidyl-Carboxy Proxyl can be synthesized using the samechemistry as described above in Example 3 for4-Succinimidyl-Carboxy-TEMPO, using3-carboxy-2,2,5,5-tetramethylpyrrolidin-1-oxyl (3-Carboxy PROXYL)instead of 4-Carboxy TEMPO as a starting material.

Example 6 Preparation of Polynitroxylated PEGylated Hemoglobin(PN-PEG-Hb)

Polynitoxylated hemoglobin was prepared in two step process: a)Preparation of PEG-conjugated hemoglobin and b) Polynitoxylation ofPEG-Hb.

PEG was conjugated to stroma-free hemoglobin (SFH) by reacting the SFHwith a 9-fold molar excess of 2-iminothiolane (2-IT) for 2.5 hours and a16-fold molar excess of Maleimide PEG 5000 (MalPEG5000) for 2 hours. Thethiolation and PEGylation reactions were performed in phosphate-bufferedsaline (PBS) at pH 7.4. As shown in the reaction scheme below, the2-iminothiolane thiolates lysine residues and the MalPEG5000 reacts withintrinsic thiols of the β-Cys93 residues and thiolated lysine residues:

R₁, R₂, and R₃ represent the remainder of the hemoglobin main chain, R₄is ethylene, and n represents the number of oxyethylene units in a 5,000dalton PEG chain. Although the reaction scheme above shows thethiolation and PEGylation as separate steps, the reaction is performedas a “one-pot” reaction, with the SFH, 2-IT, and MalPEG 5000 included ina single reaction mixture.

Polynitroxylation of the PEG-Hb was performed using carboxy-PEG-Hb. Thereaction was performed using a 30-fold molar excess of4-Succinimidyl-TEMPO-Carbonate (4-STC) over hemoglobin under anatmosphere of CO at room temperature or in refrigerated conditions. Thenumber of nitroxyl groups per hemoglobin molecule can be varied byvarying the molar excess of 4-STC over hemoglobin, the temperature atwhich the reaction is carried out, and/or reaction time. Thepolynitroxylated hemoglobin was purified using 70 kDa tangential flowfiltration and the final product was sterile filtered and stored underan atmosphere of CO. A reaction scheme for the preparation of PN-PEG-Hbfrom PEG-Hb is shown below:

R₁, R₂, and R₃ represent the remainder of the hemoglobin main chain.

Example 7 Characterization of Polynitroxylated PEGylated Hemoglobin(PN-PEG-Hb)

FIG. 4 shows Electron Paramagnetic Resonance (EPR) spectra fornon-paired electrons of TEMPOL (upper panel), and PEGylated hemoglobin(MP4) both before (middle panel) and after (lower panel)polynitroxylation (PN-MP4).

FIG. 5 presents size exclusion analysis profiles of PEGylated hemoglobin(PEG-Hb; upper panel) and polynitroxylated PEGylated hemoglobin(PEG-Hb-PN; lower panel). Size exclusion analysis was performed using aSuperose-12 column and protein was eluted using phosphate-bufferedsaline (PBS).

FIG. 6 shows a characteristic UV-Visible spectrum for polynitroxylatedPEGylated hemoglobin (PEG-Hb-PN).

The stability of the PEG-Hb-PN was tested in vivo by administering 10%top load in rats. FIG. 7 shows UV-Vis spectra of plasma hemoglobin atthe end of infusion and at one hour post infusion.

FIG. 8 shows the results of an experiment wherein PEGylated hemoglobin(MP4) was nitroxylated using a 5-, 10-, 20, 30-, 50-, or 100-fold molarexcess of 4-Succinimidyl-TEMPO-Carbonate (4-STC). The degree ofnitroxylation increased in a dose-dependent manner as the molar excessof 4-STC was increased from 5-fold to 30-fold. The degree ofnitroxylation was approximately the same when a 30-fold, 50-fold, or100-fold molar excess of 4-STC was used.

Example 8 Preparation of Polynitroxylated Albumin (PN-Alb)

Polynitroxylation of the Albumin was performed using 25% Human SerumAlbumin solution. The number of nitroxyl groups per albumin molecule wasvaried by varying the molar excess of 4-STC over albumin. The reactionwas performed using a 5-, 10-, 20-, 30-, 50 or 100-fold molar excess of4-Succinimidyl-TEMPO-Carbonate (4-STC) over albumin at pH of 7.4 for 17to 24 hours at room temperature or in refrigerated conditions. Thepolynitroxylated albumin was purified by gel filtration and analyzed byMALDI-TOF mass spectrometry to identify the number of nitroxyl groupsper albumin molecule. A reaction scheme for the preparation ofPN-Albumin from albumin using 4-Succinimidyl-TEMPO-Carbonate (4-STC) isshown below:

R₁ and R₂ represent the remainder of the albumin main chain.

The MALDI-TOF mass spectra are shown in FIGS. 9 and 13 (non-nitoxylatedHSA) and 10-12 and 14-16 (HSA nitroxylated using a 5-, 10-, 20-, 30-, 50or 100-fold molar excess of 4-STC, respectively). FIG. 17 provides agraphical representation of these data, and shows that the degree ofnitroxylation increased in a dose-dependent manner as the molar excessof 4-STC was increased from 5- to 100-fold.

Example 9 Preparation of Polynitroxylated PEGylated-Albumin (PN-PEG-Alb)

Polynitoxylated PEGylated albumin was prepared in two step process: a)Preparation of PEG-conjugated albumin and b) Polynitoxylation ofPEG-Alb.

PEG was conjugated to albumin by reacting the albumin with a 9-foldmolar excess of 2-iminothiolane (2-IT) for 2.5 hours and a 16-fold molarexcess of Maleimide PEG 5000 (MalPEG5000) for 2 hours. The thiolationand PEGylation reactions were performed in phosphate-buffered saline(PBS) at pH 7.4. After the 2 hours of PEGylation, the PEG-albuminconjugate was passed through 70 kDa tangential flow filtration to removeunreacted reagents and formulate in formulation buffer.Polynitroxylation was performed by reacting PEG-albumin with4-Succinimidyl-TEMPO-Carbonate (4-STC) using a 100-fold molar excess of4-Succinimidyl-TEMPO-Carbonate (4-STC) over albumin at room temperatureor in refrigerated conditions. The number of nitroxyl groups per albuminmolecule can be varied by varying the molar excess of 4-STC overalbumin, the temperature at which the reaction is carried out, and/orreaction time. The polynitroxylated albumin was purified using 70 kDatangential flow filtration. A reaction scheme for the preparation ofPN-PEG-Alb from PEG-Alb is shown below:

R₁, R₂, and R₃ represent the remainder of the albumin main chain, R₄ isethylene, and n represents the number of oxyethylene units in a 5,000dalton PEG chain.

What is claimed is:
 1. A nitroxylating agent of formula (I):

wherein: each of R₁, R₂, R₃, and R₄ is independently C₁-C₄ alkyl; X isoxygen, sulfur, nitrogen, phosphorus, or silicon; Y is CH₂; n is 0 or 1;and m is 0 or
 1. 2. A method for preparing the nitroxylating agent ofthe formula (II)

comprising reacting a compound having the formula (III)

with N,N′-disuccinimidyl carbonate (DSC) in the presence of an organicbase; wherein each of R₁, R₂, R₃, and R₄ is independently C₁-C₄ alkyl; Xis oxygen, sulfur, nitrogen, phosphorus, or silicon; Y is CH₂; and m is0 or
 1. 3. A method for preparing a nitroxylating agent of the formula(IV)

comprising reacting a compound having the formula (V)

with N-hydroxysuccinimide (NHS) in the presence ofN,N′-dicyclohexylcarbodiimide (DCC); wherein each of R₁, R₂, R₃, and R₄is independently C₁-C₄ alkyl; Y is CH₂; and m is 0 or
 1. 4. Anitroxylated protein having the structure (VI):

wherein Z represents the protein; each of R₁, R₂, R₃, and R₄ areindependently C₁-C₄ alkyl; X is oxygen, sulfur, nitrogen, phosphorus, orsilicon; Y is CH₂; m is 0 or 1; n is 0 or 1; p is the average number ofactivated-PEG polymers conjugated to the protein; and N is a nitrogen ofthe protein.
 5. A method for preparing a nitroxylated protein comprisingreacting the protein with a nitroxylating agent of formula (II)

wherein each of R₁, R₂, R₃, and R₄ is independently C₁-C₄ alkyl; X isoxygen, sulfur, nitrogen, phosphorus, or silicon; Y is CH₂; and m is 0or
 1. 6. A method for preparing a nitroxylated protein comprisingreacting the protein with a nitroxylating agent of formula (IV):

wherein each of R₁, R₂, R₃, and R₄ is independently C₁-C₄ alkyl; Y isCH₂; and m is 0 or
 1. 7. A nitroxylating agent, nitroxylating protein,or method of any one of claims 1-6, wherein each of R₁, R₂, R₃, and R₄is —CH₃.
 8. A nitroxylating agent of claim 1 or 7, wherein n is
 1. 9. Anitroxylating agent of claim 8, wherein X is oxygen or sulfur.
 10. Anitroxylating agent of claim 8, wherein X is oxygen.
 11. A nitroxylatingagent of claim 8, wherein X is oxygen and each of R₁, R₂, R₃, and R₄ is—CH₃.
 12. A nitroxylating agent of claim 1 or 7, wherein n is
 0. 13. Anitroxylating agent, nitroxylating protein, or method of any one ofclaims 1-12, wherein m is
 0. 14. A nitroxylating agent, nitroxylatingprotein, or method of any one of claims 1-12, wherein m is
 1. 15. Anitroxylating agent of claim 1 selected from the group consisting of:


16. A nitroxylating agent of claim 1 wherein the nitroxylating agent is:


17. A method of any one of claims 2, 7, 13 and 14, wherein the organicbase comprises triethylamine (TEA), N,N-diisopropylethylamine,4-dimethylaminopyridine, pyridine, N-methylpiperidine, or a combinationthereof.
 18. A method of claim 17, wherein the organic base comprisestriethylamine.
 19. A method of claim 18, wherein the compound of formula(III), the N,N′-disuccinimidyl carbonate, and the triethylamine arepresent in a ratio of about 1:2:3.
 20. A method of any one of claims 2,7, 13, 14 and 17-19, wherein the reaction is carried out at atemperature of about 2° C. to about 30° C.
 21. A method of claim 20,wherein the reaction is carried out at a temperature of about 15° C. toabout 25° C.
 22. A method of any one of claims 2, 7, 13, 14 and 17-21,wherein the reaction is carried out at a temperature of about 4° C. 23.A method of claim 21, wherein the reaction is carried out at atemperature of about 20° C.
 24. A method of any one of claims 2, 7, 13,14 and 17-23, wherein the reaction is allowed to proceed for about threeto about six hours.
 25. A method of any one of claims 2, 7, 13, 14 and17-24, wherein the reaction is carried out in a polar aprotic solvent.26. A method of claim 25, wherein the polar aprotic solvent comprisesacetonitrile (ACN), tetrahydrofuran (THF), ethyl acetate (EtOAc),acetone, dimethylformamide (DMF), dimethyl sulfoxide (DMSO), or acombination thereof.
 27. A method of claim 26, wherein the polar aproticsolvent comprises acetonitrile.
 28. A method of any one of claims 3, 7,13 and 14, wherein the compound of formula (IV), theN-hydroxysuccinimide, and the N,N′-dicyclohexylcarbodiimide are presentin a molar ratio of about 1:1.1:1.1.
 29. A method of any one of claims1-28, wherein m is
 0. 30. A method of any one of claims 1-28, wherein mis
 1. 31. A method of any one of claims 3, 7, 13, 14, and 28-30, whereinthe reaction is carried out at a temperature of about 2° C. to about 30°C.
 32. A method of claim 31, wherein the reaction is carried out at atemperature of about 15° C. to about 25° C.
 33. A method of claim 31,wherein the reaction is carried out at a temperature of about 4° C. 34.A method of claim 31, wherein the reaction is carried out at atemperature of about 20° C.
 35. A method of any one of claims 3, 7, 13,14, and 28-34, wherein the reaction is allowed to proceed for about 6 toabout 24 hours.
 36. A method of any one of claims 3, 7, 13, 14, and28-35, wherein the reaction is carried out at a pH of about 7.2 to about7.6.
 37. A method of any one of claims 3, 7, 13, 14, and 28-36, whereinthe reaction is carried out at a pH of about 7.4.
 38. A nitroxylatedprotein of any one of claims 4, 7, 13, 14, 29, and 30, wherein theN-terminal amino group of the protein is nitroxylated.
 39. Anitroxylated protein of any one of claims 4, 7, 13, 14, 29, and 30,wherein at least one epsilon (ε)-amino group of a lysine residue isnitroxylated.
 40. A nitroxylated protein of any one of claims 4, 7, 13,14, 29, 30, 38 and 39, wherein p is about 1 to about
 25. 41. Anitroxylated protein of any one of claims 4, 7, 13, 14, 29, 30, 38 and39, wherein p is at least about
 2. 42. A nitroxylated protein of claim41, wherein p is at least about
 10. 43. A nitroxylated protein of claim40, wherein p is about 15 to about
 20. 44. A nitroxylated protein of anyone of claims 4, 7, 13, 14, 29, 30, and 38-43, wherein the proteincomprises a hemoglobin (Hb) α-subunit, a hemoglobin β-subunit, ahemoglobin tetramer, a myoglobin, or an albumin.
 45. A nitroxylatedprotein of claim 44, wherein the protein comprises a serum albumin. 46.A nitroxylated protein of claim 45, wherein the serum albumin compriseshuman serum albumin (HSA).
 47. A nitroxylated protein of claim 44,wherein the protein comprises a hemoglobin α-subunit or a hemoglobinβ-subunit or hemoglobin tetramer.
 48. A nitroxylated protein of claim44, wherein the protein comprises a human hemoglobin α-subunit, an humanhemoglobin β-subunit, or a hemoglobin tetramer comprising humanhemoglobin α-subunits and β-subunits.
 49. A nitroxylated protein of anyone of claim 44, 47 or 48, wherein the hemoglobin tetramer comprises across-linked αα dimer or a cross-linked ββ dimer.
 50. A nitroxylatedprotein of claim 49, wherein the nitroxylated protein comprises a humanhemoglobin α-subunit.
 51. The nitroxylated protein of claim 50, whereinthe human hemoglobin α-subunit is nitroxylated at the α-amino group ofthe N-terminal valine residue.
 52. The nitroxylated protein of claim 50or 51, wherein the human hemoglobin α-subunit is nitroxylated at theε-amino group of a lysine residue selected from the group consisting oflysine-7, lysine-11, lysine-16, lysine-40, lysine-56, lysine-60,lysine-61, lysine-90, lysine-99, lysine-127, lysine-139, and acombination thereof.
 53. A nitroxylated protein of claim 50, wherein thenitroxylated protein comprises a human hemoglobin β-subunit.
 54. Anitroxylated protein of claim 53, wherein the human hemoglobin β-subunitis nitroxylated at the α-amino group of the N-terminal valine residue.55. A nitroxylated protein of claim 53 or 54, wherein the humanhemoglobin β-subunit is nitroxylated at the ε-amino group of a lysineresidue selected from the group consisting of lysine-8, lysine-17,lysine-59, lysine-61, lysine-65, lysine-66, lysine-82, lysine-95,lysine-120, lysine-132, lysine-144, and a combination thereof.
 56. Anitroxylated protein of any one claims 44-55, wherein the proteincomprises hemoglobin tetramer and the hemoglobin tetramer comprisesabout seventeen nitroxylated amino groups.
 57. A nitroxylated protein ofany one of claims 4, 7, 13, 14, 29, 30 and 38-56, wherein thenitroxylated protein is conjugated to a polyalkylene oxide (PAO).
 58. Anitroxylated protein of claim 57, wherein the PAO is a polyethyleneglycol (PEG).
 59. A nitroxylated protein of claim 58, wherein the PEGhas an average molecular weight of about 2,000 to about 20,000 Daltons.60. A nitroxylated protein of claim 58, wherein the PEG has an averagemolecular weight of about 3,000 to about 10,000 Daltons.
 61. Anitroxylated protein of claim 58, wherein the PEG has an averagemolecular weight of about 4,000 to about 6,000 Daltons.
 62. Anitroxylated protein of claim 61, wherein the PEG has an averagemolecular weight of about 5,000 Daltons.
 63. A nitroxylated protein ofany one of claims 58-62, wherein the PEG is a maleimide-PEG.
 64. Anitroxylated protein of claim 63, wherein the maleimide is linked to thePEG via an alkylene or phenylene linker.
 65. A nitroxylated protein ofclaim 64, wherein the alkylene linker is an ethylene linker.
 66. Anitroxylated protein of any one of claims 63-65, wherein themaleimide-PEG is conjugated to a thiol moiety of the protein selectedfrom the group consisting of an intrinsic thiol moiety of a cysteineresidue of the protein, a thiol moiety of a thiolated lysine residue ofthe protein, and a combination thereof.
 67. A nitroxylated protein ofclaim 66, wherein the maleimide-PEG conjugated to an intrinsic thiolmoiety of a cysteine residue or conjugated to a thiol moiety of athiolated lysine residue has the structure (VIII)

wherein Z represents the protein, S is a thiol of the protein, R₃ is analkylene or phenylene group, X is a terminal group, m is the averagenumber of activated-PEG polymers conjugated to the protein, and nrepresents the average number of oxyethylene units of a PEG having anaverage molecular weight of about 2,000 to about 20,000 Daltons.
 68. Anitroxylated protein of claim 67, wherein R₃ is ethylene.
 69. Anitroxylated protein of claim 67 or 68, wherein m is about 6 to about10.
 70. A nitroxylated protein of any one of claims 67-69, wherein X ismethoxy (—OCH₃) or carboxylate (—COOH).
 71. A nitroxylated protein ofany one of claims 63-70, wherein the maleimide-PEG is conjugated to athiol moiety of a cysteine-93 residue of a hemoglobin β-subunit.
 72. Anitroxylated protein of any one of claims 63-71, wherein themaleimide-PEG is conjugated to a thiol moiety of a thiolated lysineresidue of a hemoglobin α-subunit or β-subunit.
 73. A nitroxylatedprotein of claim 72, wherein the thiolated lysine residue is a thiolatedlysine residue of a human hemoglobin α-subunit selected from the groupconsisting of lysine-7, lysine-11, lysine-16, lysine-40, lysine-56,lysine-60, lysine-61, lysine-90, lysine-99, lysine-127, lysine-139, anda combination thereof.
 74. A nitroxylated protein of claim 72, whereinthe thiolated lysine residue is a thiolated lysine residue of a humanhemoglobin β-subunit selected from the group consisting of lysine-8,lysine-17, lysine-59, lysine-61, lysine-65, lysine-66, lysine-82,lysine-95, lysine-120, lysine-132, lysine-144, and a combinationthereof.
 75. A nitroxylated protein of any one of claims 58-62, whereinthe PEG is a succinimidyl valerate PEG (SVA-PEG).
 76. A nitroxylatedprotein of claim 75, wherein the SVA-PEG is conjugated to an aminomoiety of the protein selected from an ε-amino moiety of a lysineresidue of the protein, an α-amino moiety of a terminal valine residueof the protein, or a combination thereof.
 77. A nitroxylated protein ofclaim 76, wherein the SVA-PEG conjugated to an ε-amino moiety of alysine residue of the protein or an α-amino moiety of a terminal valineresidue of the protein has the structure (IX)

wherein: Z is the protein, N is an amino group of the protein, X is aterminal group, m is the number of activated-PEG polymers conjugated tothe protein, and n is the average number of oxyethylene units of a PEGhaving an average molecular weight of from about 2,000 to about 20,000Daltons.
 78. A nitroxylated protein of claim 77, wherein X is methoxy(—OCH₃) or carboxylate (—COOH).
 79. A nitroxylated protein of claim 77or 78, wherein m is on average from about 6 to about 10 PAO moleculesper tetramer.
 80. A nitroxylated protein of any one of claims 75-79,wherein the SVA-PEG is conjugated to an ε-amino moiety of a lysineresidue of a hemoglobin α-subunit or β-subunit.
 81. A nitroxylatedprotein of any one of claims 75-80, wherein the SVA-PEG is conjugated toan α-amino moiety of a terminal valine residue of a hemoglobin α-subunitor β-subunit.
 82. A nitroxylated protein of claim 80 or 81, wherein thelysine residue is a lysine residue of a human hemoglobin α-subunitselected from the group consisting of lysine-7, lysine-11, lysine-16,lysine-40, lysine-56, lysine-60, lysine-61, lysine-90, lysine-99,lysine-127, lysine-139, and a combination thereof.
 83. A nitroxylatedprotein of claim 80 or 81, wherein the lysine residue is a lysineresidue of a human hemoglobin β-subunit selected from the groupconsisting of lysine-8, lysine-17, lysine-59, lysine-61, lysine-65,lysine-66, lysine-82, lysine-95, lysine-120, lysine-132, lysine-144, anda combination thereof.
 84. A hemoglobin tetramer comprising at least oneα-subunit of any one of claims 44, 47, 48, 50-52, 57-70, 72, 73, and75-82 or at least one β-subunit of any one of claims 44, 47, 48, 53-55,57-72, 74-81, and
 83. 85. The hemoglobin tetramer of claim 84,comprising at least one α-subunit of any one of claims 44, 47, 48,50-52, 57-70, 72, 73, and 75-82 and at least one β-subunit of any one ofclaims 44, 47, 48, 53-55, 57-72, 74-81, and
 83. 86. The hemoglobintetramer of claim 85, comprising two α-subunits of any one of claims 44,47, 48, 50-52, 57-70, 72, 73, and 75-82 and two β-subunits of any one ofclaims 44, 47, 48, 53-55, 57-72, 74-81, and
 83. 87. A hemoglobintetramer of any one of claims 84-86, wherein the hemoglobin isconjugated to on average 5 to 10 PAO molecules per tetramer.
 88. Ahemoglobin tetramer of claim 87, wherein the hemoglobin is conjugated toon average 7.1 to 8.9 PAO molecules per tetramer.
 89. A hemoglobintetramer of any one of claims 84-88, wherein the hemoglobin isoxygenated.
 90. A hemoglobin tetramer of any one of claims 84-89,wherein the hemoglobin is deoxygenated.
 91. A hemoglobin tetramer of anyone of claims 84-90, wherein the hemoglobin is liganded with CO, NO, ora mixture of CO and NO.
 92. A method of any one of claims 5-7, 13, 14,29, and 30, wherein the ratio of the nitroxylating agent is present atabout a 5-fold to about 100-fold molar excess over the protein.
 93. Amethod of any one of claims 5-7, 13, 14, 29, 30 and 92, wherein theprotein comprises an α- or β-subunit of a hemoglobin tetramer.
 94. Amethod of any one of claims 5-7, 13, 14, 29, 30, 92 and 93, wherein theprotein comprises a hemoglobin tetramer.
 95. A method of claim 94,wherein the hemoglobin tetramer is a non-oxygenated hemoglobin tetramer.96. A method of claim 95, wherein the non-oxygenated hemoglobin tetrameris a CO-liganded hemoglobin tetramer.
 97. A method of claim 95, whereinthe hemoglobin tetramer is a deoxygenated hemoglobin tetramer.
 98. Amethod of any one of claims 5-7, 13, 14, 29, 30, 92-97, wherein thereaction is carried out at a temperature of about 2° C. to about 30° C.99. A method of claim 98, wherein the reaction is carried out at atemperature of about 15° C. to about 25° C.
 100. A method of claim 98,wherein the reaction is carried out at about 2° C. to about 8° C.
 101. Amethod of claim 98, wherein the reaction is carried out at a temperatureof about 4° C.
 102. A method of claim 98, wherein the reaction iscarried out at a temperature of about 20° C.
 103. A method of any one ofclaims 5-7, 13, 14, 29, 30, 92-102, wherein the reaction is allowed toproceed for about three to about 20 hours.
 104. A method of claim 103,wherein the reaction is allowed to proceed for about three to about sixhours.
 105. A method of claim 103, wherein the reaction is allowed toproceed for about 16 hours.
 106. A method of any one of claims 5-7, 13,14, 29, 30, 92-105, wherein the reaction is carried out in an aqueoussolvent.
 107. A method of any one of claims 5-7, 13, 14, 29, 30, 92-106,wherein the reaction is carried out at a pH of about 6.5 to about 8.5.108. A method of claim 107, wherein the reaction is carried out at a pHof about 7.5.
 109. A method of claim 107, wherein the reaction iscarried out at a pH of about 7.2.
 110. A method of any one of claims5-7, 13, 14, 29, 30, 92-109, wherein the protein comprises a hemoglobintetramer, the reaction is carried out at a pH of about 7.2 and atemperature of about 2° C. to about 8° C. and is allowed to proceed forabout sixteen hours, and wherein the method yields a nitroxylatedhemoglobin tetramer having about 17 nitroxylated amino groups.
 111. Amethod of any one of claims 5-7, 13, 14, 29, 30, and 92-110, wherein theprotein comprises a hemoglobin tetramer, and the nitroxylating agent ispresent at about a 10- to about 100-fold molar excess over thehemoglobin tetramer.
 112. A method of any one of claims 5-7, 13, 14, 29,30, and 92-111, wherein the product of the reaction is a nitroxylatedprotein of any one of claims 4, 7, 13, 14, 29, 30 and 38-83 or ahemoglobin tetramer of any one of claims 84-91.
 113. A method of any oneof claims 5-7, 13, 14, 29, 30, and 92-112, further comprisingconjugating the protein to a polyalkylene oxide (PAO).
 114. A method ofclaim 113, further comprising: adding succinimidyl valerate PAO to theprotein in the aqueous diluent to form a PAO-valerate conjugatedprotein.
 115. A method of claim 113, further comprising: mixing theprotein with 2-iminothiolane (2-IT) in an aqueous diluent to form athiolated protein; and adding PAO-maleimide to the thiolated protein inthe aqueous diluent to form a PAO-maleimide conjugated protein.
 116. Amethod of any one of claims 113-115, wherein the protein comprises ahemoglobin tetramer.
 117. A method of claim 113 or 116, wherein thehemoglobin tetramer comprises a cross-linked αα dimer or a cross-linkedββ dimer.
 118. The method of any one of claims 115-117, wherein the2-iminithiolane is present at a concentration of between about 7- andabout 15-fold molar excess over the protein concentration.
 119. Themethod of any one of claims 115-117, wherein the 2-iminothiolane ispresent at a concentration of between about 7- and about 8-fold molarexcess over the protein concentration.
 120. The method of any one ofclaims 115-117, wherein the 2-iminothiolane is present at aconcentration of between about 7-0.5-fold molar excess over the proteinconcentration.
 121. The method of any one of claims 115-120, wherein thePAO-maleimide is present at a concentration of between about 9- andabout 20-fold molar excess over the protein concentration.
 122. Themethod of any one of claims 115-120, wherein the PAO-maleimide ispresent at a concentration of between about 9- and about 15-fold molarexcess over the protein concentration.
 123. The method of any one ofclaims 115-120, wherein the PAO-maleimide is present at a concentrationof between about 12-fold molar excess over the protein concentration.124. The method of any one of claims 115-123, wherein the thiolationstep is carried out at a pH of between about 7 and about
 9. 125. Themethod of any one of claims 115-123, wherein the thiolation step iscarried out at a pH of about 8.5.
 126. The method of any one of claims115-125, wherein the step of adding the PAO-maleimide to the thiolatedprotein to form a PAO-maleimide conjugated protein is carried out at apH of between about 6.5 and about 8.5.
 127. The method of any one ofclaims 115-125, wherein the step of adding the PAO-maleimide to thethiolated protein to form a PAO-maleimide conjugated protein is carriedout at a pH of about 7.5.
 128. The method of any one of claims 113-127,wherein the protein is conjugated to PAO prior to nitroxylation of theprotein.
 129. The method of claim 115-128, wherein the step of addingthe PAO-maleimide to the thiolated protein to form a PAO-maleimideconjugated protein is performed concurrently with nitroxylation of theprotein.
 130. The method of claim 114-129, wherein the step of addingthe succinimidyl valerate PAO to the protein to form a PAO-valerateconjugated protein is performed concurrently with nitroxylation of theprotein.
 131. A pharmaceutical composition comprising a nitroxylatedprotein of any one of claims 4, 7, 13, 14, 29, 30, and 38-83 or ahemoglobin tetramer of any one of claims 84-91 and a pharmaceuticallyacceptable carrier.
 132. A pharmaceutical composition of claim 131,wherein the composition is normooncotic with blood.
 133. Apharmaceutical composition of claim 131 or 132, wherein the compositionis hyperoncotic as compared to blood.
 134. A pharmaceutical compositionof any one of claims 131-133, wherein the pharmaceutically acceptablecarrier comprises an aqueous diluent.
 135. A pharmaceutical compositionof claim 134, wherein the aqueous diluent comprises an aqueous solutionof a colloid or an aqueous solution of a non-oxygen carrying component.136. A pharmaceutical composition of claim 134 or 135, wherein theaqueous diluent comprises an aqueous cell-free solution.
 137. Apharmaceutical composition of any one of claims 134-136, wherein theaqueous diluent comprises an aqueous solution of proteins, an aqueoussolution of glycoproteins, an aqueous solution of polysaccharides, or acombination thereof.
 138. A pharmaceutical composition of any one of134-137, wherein the aqueous diluent comprises an aqueous cell-freesolution of albumin.
 139. A pharmaceutical composition of any one ofclaims 131-138, wherein the pharmaceutically acceptable carriercomprises physiological saline, a saline-glucose mixture, Ringer'ssolution, lactated Ringer's solution, Locke-Ringer's solution,Krebs-Ringer's solution, Hartmann's balanced saline, heparinized sodiumcitrate-citric acid-dextrose solution, an acetate solution, a multipleelectrolyte solution, a lactiobionate solution, a polymeric plasmasubstitute, or a combination thereof.
 140. A pharmaceutical compositionof claim 139, wherein the polymeric plasma substitute comprisespolyethylene oxide, polyvinyl pyrrolidone, polyvinyl alcohol, anethylene oxide-propylene glycol condensate, or a combination thereof.141. The pharmaceutical composition of any one of claims 131-140,wherein the pharmaceutically acceptable carrier comprises a filler, asalt, a physiological buffer, a carbohydrate, an alcohol, a polyalcohol, an anti-oxidant, an anti-bacterial agent, an oncotic pressureagent, a reducing agent, or a combination thereof.
 142. Thepharmaceutical composition of claim 141, wherein the reducing agentcomprises, ascorbic acid, glutathione, N-acetyl cysteine, or acombination thereof.
 143. A pharmaceutical composition of any one ofclaims 131-142, for use in the treatment of acute liver failure, betathalassemia, a burn, chronic critical limb ischemia, carbon dioxide orcyanide poisoning, chronic obstructive pulmonary disease (COPD),congestive heart failure, hypoxia, malaria, organ ischemia, peripheralvascular disease, porphyria, pre-eclampsia in pregnancy, sepsis, sicklecell disease, retinal disease, an intra-ocular condition, testiculartorsion, trauma, shock, traumatic brain injury, ulcers, vasospasm, or acombination thereof.
 144. A pharmaceutical composition of claim 143,wherein the organ ischemia comprises acute bowel ischemia (torsion),acute bowel ischemia (embolism), cardiogenic shock, acute vascular organischemia, stroke, myocardial infarction, or severe cardiac ischemia.145. A pharmaceutical composition of any one of claims 131-142, for usein the treatment of non-traumatic hemorrhagic shock, pre-hospitalsetting trauma, traumatic hemorrhagic shock, acute lung injury, adultrespiratory distress syndrome, traumatic brain injury, stroke, solidtumor cancer, organ degradation (ex-vivo), organ degradation (inrecipient), severe sepsis, septic shock, myocardial infarction, cardiacischemia, cardiogenic shock, acute heart failure, pulmonary embolism, ora combination thereof.
 146. A pharmaceutical composition of any one ofclaims 131-142, for use as an adjunct to angioplasty, as an adjunct forplastic surgery, or as an adjunct in implanting a ventricular assistdevice; as a blood substitute, a cardioprotectant, a cryopreservative, ahemodialysis adjunct, an oncology agent, an organ preservative, aperformance enhancement agent, a surgery adjunct, or a wound healingagent; in imaging; to improve lung function; or a combination thereof.147. A pharmaceutical composition of any one of claims 131-142, forveterinary treatment of of loss of blood due to injury, hemolyticanemia, infectious anemia, bacterial infection, Factor IV fragmentation,hypersplenation and splenomegaly, hemorrhagic syndrome in poultry,hypoplastic anemia, aplastic anemia, idiopathic immune hemolyticconditions, iron deficiency, isoimmune hemolytic anemia,microangiopathic hemolytic anemia, parasitism, or surgical-anesthesiainduced brain damage.
 148. A method of treatment comprisingadministering a hemoglobin tetramer of any one of claims 84-91 or apharmaceutical composition of any one of claims 131-147 to a subject inneed thereof.
 149. A method of treatment of claim 148, wherein thesubject is an animal.
 150. A method of treatment of claim 149, whereinthe subject is a human.
 151. A method of treatment of any one of claims148-150, wherein the method is a method for the treatment of acute liverfailure, beta thalassemia, a burn, chronic critical limb ischemia,carbon dioxide or cyanide poisoning, chronic obstructive pulmonarydisease (COPD), congestive heart failure, hypoxia, malaria, organischemia, peripheral vascular disease, porphyria, pre-eclampsia inpregnancy, sepsis, sickle cell disease, retinal disease, an intra-ocularcondition, testicular torsion, trauma, shock, traumatic brain injury,ulcers, vasospasm, or a combination thereof.
 152. The method oftreatment of claim 151, wherein the organ ischemia comprises acute bowelischemia (torsion), acute bowel ischemia (embolism), cardiogenic shock,acute vascular organ ischemia, stroke, myocardial infarction, or severecardiac ischemia.
 153. A method of treatment of any one of claims148-150, wherein the method is a method for the treatment ofnon-traumatic hemorrhagic shock, pre-hospital setting trauma, traumatichemorrhagic shock, acute lung injury, adult respiratory distresssyndrome, traumatic brain injury, stroke, solid tumor cancer, organdegradation (ex-vivo), organ degradation (in recipient), severe sepsis,septic shock, myocardial infarction, cardiac ischemia, cardiogenicshock, acute heart failure, pulmonary embolism, or a combinationthereof.
 154. A method of treatment of any one of claims 148-150,wherein the hemoglobin tetramer or pharmaceutical composition isadministered as an adjunct to angioplasty, as an adjunct for plasticsurgery, or as an adjunct in implanting a ventricular assist device; asa blood substitute, a cardioprotectant, a cryopreservative, ahemodialysis adjunct, an oncology agent, an organ preservative, aperformance enhancement agent, a surgery adjunct, or a wound healingagent; in imaging; to improve lung function; or a combination thereof.155. A method of treatment of any one of claims 148-150, wherein thehemoglobin tetramer or pharmaceutical composition is administered as anadjunct to angioplasty, as an adjunct to thoracic aortic repairs, as anadjunct to cardiopulmonary bypass, or as a priming solution forcardiopulmonary bypass.
 156. A method of treatment of claim 148 or 149,wherein the subject is a non-human animal and the method is a method forveterinary treatment of loss of blood due to injury, hemolytic anemia,infectious anemia, bacterial infection, Factor IV fragmentation,hypersplenation and splenomegaly, hemorrhagic syndrome in poultry,hypoplastic anemia, aplastic anemia, idiopathic immune hemolyticconditions, iron deficiency, isoimmune hemolytic anemia,microangiopathic hemolytic anemia, parasitism, or surgical-anesthesiainduced brain damage.
 157. A method of delivering oxygen, nitric oxide,carbon monoxide or mixtures thereof to tissue and reducing nitrite tonitric oxide (NO) in the microvasculature, the method comprisingadministering the nitroxylated protein of any one of claims 4, 7, 13,14, 29, 30 and 38-83 or the pharmaceutical composition of any one ofclaims 131-147 to a subject in need thereof, wherein followingadministration, the hemoglobin becomes unliganded and converts nitriteto nitric oxide in the microvasculature.